STANDARD MEASUREMENTS, DATA, Common Abbreviations

APPENDIX 1STANDARD MEASUREMENTS, DATA,AND ABBREVIATIONS

APPENDIX 1ACommon AbbreviationsA260 absorbance at 260 nmA adenine or adenosine; one-letter code for

alanineAb antibodyABTS [2,2′-azino-di(3-ethylbenzothiazoline

sulfonate)]acetyl CoA acetyl coenzyme AAcMNPV Autographica californica

multiply-enveloped nuclear polyhedrosisvirus

ADA adenosine deaminaseADC analog-to-digital converterADH alcohol dehydrogenaseADP adenosine 5′-diphosphateADSL asynchronous digital subscriber lineAEC 3-amino-9-ethylcarbazoleAES 3-aminopropyltriethoxysilaneAEX anion exchangeAFLP amplified fragment length

polymorphismAg antigenAIDS acquired immune deficiency syndromeAK adenosine kinaseALARA as low as reasonably achievableALPS autoimmune/lymphoproliferative

syndromeAM acetomethyl (moiety)AMan anhydro-D-mannoseAMP adenosine 5′-monophosphateAMPPD disodium

3,4-methoxyspiro(1,2-dioxetane-3,2-tricyclo[3.3.1.13,7]decan)phenylphosphate

AMV avian myeloblastosis virusANOVA analysis of varianceAP alkaline phosphatase; apyrimidinic

(sites)APH aminoglycoside phosphotransferaseAPHIS Animal and Plant Health Inspection

ServiceaPKC atypical protein kinase CApr ampicillin resistantAPRT adenosine phosphoribosyltransferaseAPS ammonium persulfateARS autonomous replication sequencesASPECT augmented surface polyethylene

prepared by chemical transformationATA aurintricarboxylic acidATCC American Type Culture CollectionATP adenosine 5′-triphosphate

AUFS absorbance units, full scaleAUS Arthrobacter ureafaciensββ-gal β-galactosidaseBAC bacterial artificial chromosome;

biospecific affinity chromatographyBAP bacterial alkaline phosphataseBBS BES-buffered solution; borate-buffered

salineBCIP 5-bromo-4-chloro-3-indolyl phosphateBDB bis-diazobenzidineBES N,N-bis(2-hydroxyethyl)-2-

aminoethanesulfonic acidBHI brain heart infusion (medium)biotin-11-dUTP 8-(2,4-dinitrophenyl-2,6-

aminohexyl)aminoadenosine-5′-triphosphate or 2′-deoxyuridine-5′-triphosphate-5′-allylamin biotin

bis; bisacrylamide N,N′-methylenebisacrylamide

bis-Tris 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol

BLAST Basic Local Alignment ResearchTool

Bluo-gal indoyl-β-D-galactopyranosideBMP bitmap (file format)Boc t-butyloxycarbonylBOP benzotriazolyl-N-oxy-

tris(dimethylamino)phosphoniumhexafluorophosphate

bp base pairBPV bovine papilloma virusBq BecquerelBrdU 5-bromodeoxyuridineBS3 bis(sulfosuccinimidyl) suberateBSA bovine serum albuminBSL biosafety levelBst Bacillus stearothermophilus DNA

(polymerase)C cytosine or cytidine; one-letter code for

cysteineC16TAB hexadecyl trimethylammonium

bromideCA3 chromomycin A3CAD carbamoylphosphate synthetaseCaM calmodulincAMP adenosine 3′,5′-cyclic-monophosphatecA-PrK cyclic AMP-dependent protein

kinaseCAPS [cyclohexylamino]-1-propanesulfonic

acid

Current Protocols in Molecular Biology (2005) A.1A.1-A.1A.8Copyright C© 2005 by John Wiley and Sons, Inc.

StandardMeasurements,Data, andAbbreviations

A.1A.1

Supplement 70

CommonAbbreviations

A.1A.2

Supplement 70 Current Protocols in Molecular Biology

CAT chloramphenicol acetyltransferaseCATH Class, Architecture, Topology, and

Homologous superfamilyCCD charge-coupled deviceCCR Coriell Cell RepositoryCD cluster of differentiation (antigens);

circular dichroismCDC Centers for Disease ControlcDNA complementary deoxyribonucleic

acidCD-ORD circular dichroism–optical

rotatory dispersionCDP cytidine 5′-diphosphateCD-ROM compact disk read-only memoryCDS coding sequenceCE capillary electrophoresisCED 3′ cyanoethyl protectedCEF chicken embryo fibroblastCERN European Nuclear Research CouncilCEX cation exchangeCFA complete Freund’s adjuvantCFU colony-forming unitCGH comparative genome hybridizationCHAPS 3-[(3-cholamidopropyl)-

dimethylammonio]-1-propane-sulfonateCHEF contour-clamped homogeneous

electric fieldCHES 2-(N-

cyclohexylamino)ethanesulfonicacid

CHO Chinese hamster ovary (cells)Ci curieCID charge-injection device;

collision-induced dissociationCIP calf intestine phosphataseCLEAR cross-linked acrylate ethoxylate

resincM centimorgansCM complete minimal (medium);

carboxymethylCMC critical micelle concentrationCML chronic myelogenous leukemiaCMP cytidine 5′-monophosphateCmr chloramphenicol resistantCMV cytomegalovirus4CN 4-chloro-1-naptholCNBr cyanogen bromideCon A concanavalin ACORT cloning of receptor targetsCPC cetylpyridinium chlorideCPG control pore glasscpm counts per minuteCRD cross-reacting determinantCS chemical sequencingCSPD disodium 3-(4-methoxyspiro[1,2-

dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.13,7]decan]-4-yl)phenyl phosphate

CTAB cetyltrimethylammonium bromide

CTC charge-transfer chromatographyCTD C-terminal domainCTP cytidine 5′-triphosphateCWS cell wall skeletonCXA contextual expression analysisD dextrorotatorydA deoxyadenosineDa DaltonDAB 3,3′-diaminobenzidineDABCO 1,4-diazobicyclo-[2.2.2]octaneDAD diode array detectionDAG diacylglyceroldAMP deoxyadenosine monophosphateDAPI 4′,6-diamidino-2-phenylindoled(A-T) deoxyadenylate-deoxythymidylatedATP deoxyadenosine triphosphateDBE direct blotting electrophoresisDBM diazobenzyloxymethyldC deoxycytosineDCA dichloroacetic acidDCC dextran-coated charcoal;

N,N′-dicyclohexylcarbodiimidedCF 2′-deoxycoformycindCMP deoxycytidine monophosphatedCTP deoxycytidine triphosphateDD differential displayddATP dideoxyadenosine triphosphateDDBJ DNA Data Bank of JapanddCTP dideoxycytidine triphosphateDDDS distributed document delivery systemddGTP dideoxyguanosine triphosphateddNTP dideoxynucleoside triphosphateddTTP dideoxythymidine triphosphateDEA diethylamineDEAE diethylaminoethylDEPC diethylpyrocarbonateDES diethylstilbestroldf degrees of freedomdG deoxyguanosinedGTP deoxyguanosine triphosphateDHFR dihydrofolate reductaseDIEA N,N-diisopropylethylamineDIGE difference gel electrophoresisDiI 1,1′-dihexyl-3,3,3′,3′-

tetramethylindocarbocyanine perchlorateDIP Database of Interacting Proteins;

deletion-insertion polymorphismdITP deoxyinosine 5′-triphosphateDMB 1,2-diamino-4,5-

methylenedioxybenzenedihydrochloride

DMEM Dulbecco’s modified Eagle (orminimum essential) medium

DMF dimethylformamideDMS dimethyl sulfateDMSO dimethyl sulfoxideDMT dimethoxytritylDNA deoxyribonucleic acid


A.1A.3

Current Protocols in Molecular Biology Supplement 70

DNase deoxyribonucleaseDNP 2,4-dinitrophenyldNTP deoxynucleoside triphosphateDOTMA n-[1-(2,3-dioleoyloxy)propyl]-

N,N,N,-trimethylammonium chlorideDPA diphenylaminedpm disintegrations per minuteds double-strandedDSA Datura stramonium agglutininDSG disuccinimidyl glutarateDSL digital subscriber lineDSS disuccinimidyl suberatedT deoxythymidineDTE direct transfer electrophoresisDTT dithiothreitoldTTP deoxythymidine triphosphatedUMP deoxyuridine monophosphatedUTP deoxyuridine triphosphateDvB deleted V (Mls-like loci)EBI European Biotechnology InformationEBV Epstein-Barr virusEC embryonic carcinoma; Enzyme

CommissionECL enhanced chemiluminescenceECTEOLA epichlorohydrin triethanolamineEDC N-ethyl-N′-[(dimethylamino) propyl]

carbodiimide hydrochlorideEDTA ethylenediaminetetraacetic acidEGCase endoglycoceramidaseEGFR epidermal growth factor receptorEGTA ethylene glycol-bis(β-aminoethyl

ether)-N,N,N′,N′-tetraacetic acidELISA enzyme-linked immunosorbent assayEMBL European Molecular Biology

LaboratoryEMC encephalomyocarditisEMCV encephalomyocarditis virusEMS ethyl methanesulfonateEMSA electrophoretic mobility shift assayEndo endoglycosidaseEOF electroosmotic flowEP-PCR error-prone polymerase chain

reactionES embryonic stem (cells)ESI electrospray ionization (mass

spectrometry)EST expressed sequence tagET energy transferexo exonucleaseF FaradFACS fluorescence-activated cell sortingFAQ frequently asked questionsFBS fetal bovine serumFCS fetal calf serumFITC fluorescein isothiocyanateFFPE formalin-fixed paraffin-embedded

(tissue)FIGE field-inversion gel electrophoresis

FISH fluorescence in situ hybridizationFmoc fluorenylmethyloxycarbonylFOA fluoroorotic acidFPLC fast protein; fast peptide; or fast

polynucleotide liquid chromatographyFQDN fully-qualified domain nameFRET fluorescent resonant energy transferFSC forward (light) scatter (in flow

cytometry)FSSP Fold classification based on

Structure-Structure alignment of ProteinsFTIR Fourier transform infrared

(spectroscopy)FTP File Transfer ProtocolFuc L-fucoseFUdR 5-fluoro-2′-deoxyuridineg gravity (unit of centrifugal force)G gauge; guanine or guanosine; one-letter

code for glycineGAG glycosaminoglycanGal D-galactoseGalNAc N-acetylgalactosamineGALV gibbon ape leukemia virusGANC gancyclovirGb gigabyteGDB Genetic Data BaseGDP guanosine 5′-diphosphateGEM gene expression monitoringGF gel filtration (chromatography)GFP green fluorescent proteinGIF graphics interchange (file) formatGlc D-glucoseGlcA D-glucuronic acidGLC-FID gas-liquid chromatography with

flame ionization detectionGLC-MS gas-liquid chromatography with

mass spectroscopic detectionGlcN D-glucosamineGlcNAc N-acetylglucosamineGlUA; GlA D-glucuronic acidGM-CSF granulocyte/macrophage colony

stimulating factorGMP guanosine monophosphateGMS genomic mismatch scanningGNA Galanthus nivalus agglutininGPI glycosyl phosphatidyl inositolGRAIL Gene Recognition and Analysis

Internet LinkGRASS Graphical Representation and

Analysis of Structure ServerGS glutamine synthetaseGSS genome survey sequenceGST glutathione S-transferasegTLD generic top-level domainsGTP guanosine 5′-triphosphateGUI graphical user interfaceGUS β-glucuronidaseGy Gray (radioactivity unit)

CommonAbbreviations

A.1A.4


HA (influenza) hemagglutinin proteinHAT hypoxanthine/aminopterin/thymidine

(medium)HATU O-(7-azabenzotriazol-1-yl)-

N,N,N′,N′-tetramethyluroniumhexafluorophosphate

HBSS Hanks’ buffered salt solutionHBTU O-benzotriazol-1-yl-N,N,N′,N′-

tetramethyluronium hexafluorophosphateHCG human chorionic gonadotropinhCMV human cytomegalovirusHeBS HEPES-buffered salineHEC hydroxyethylcelluloseHEPA high-efficiency particulate air (filter)HEPES N-2-hydroxyethylpiperazine-N′-2-

ethanesulfonic acidHFBA heptafluorobutyric acidhGH human growth hormoneHGPRT hypoxanthine-guanine

phosphoribosyltransferaseHIC hydrophobic interaction

chromatographyHILIC hydrophilic interaction

chromatographyHIV human immunodeficiency virusHOAt 1-hydroxy-7-azabenzotriazoleHOBt 1-hydroxybenzotriazoleHPAE-PAD high-performance anion

exchange chromatography with pulsedamperometric detection

HP-BAC high-performance biospecific/biomimetic affinity chromatography

HPCF high-performance chromatofocusingHP-CTC high-performance charge transfer

chromatographyHPH hygromycin-B-phosphotransferaseHP-HIC high-performance

hydrophobic-interaction chromatographyHP-HILIC high-performance hydrophilic

interaction chromatographyHP-IEX high-performance ion-exchange

chromatographyHP-IMAC high-performance immobilized

metal ion affinity chromatographyHPLC high-performance liquid

chromatographyHP-LEC high-performance ligand-exchange

chromatographyHPMC hydroxypropyl methyl celluloseHP-MMC high-performance mixed mode

chromatographyHP-NPC high-performance normal phase

chromatographyHPRT hypoxathine-guanine

phosphoribosyltransferaseHP-SEC high-performance size-exclusion

chromatography

HRPO horseradish peroxidasehsiRNA heterochromatic short interfering

RNAHS-TBST high-salt TBST (buffer)HSV herpes simplex virusHTG/HTGS high-throughput genome

sequenceHTML hypertext markup languageHz hertzIAA 3-β indoleacrylic acid; indole-3-acetic

acidIACUC Institutional Animal Care and Use

CommitteeICAT isotope coded affinity taggingi.d. inner diameterIdoA; IdUA; IdA L-iduronic acidIEF isoelectric focusingIEX ion exchangeIg immunoglobulinimm immunity regionIMAC immobilized metal affinity

chromatographyIMPDH inosine-monophosphate

dehydrogenaseIODOGEN 1,3,4,6-tetrachloro-3α,6α-

diphenylglycourilIP Internet ProtocolIPTG isopropyl-1-thio-β-D-galactosideIR infraredIRES internal ribosomal entry siteISDN integrated services digital networkISH in situ hybridizationISP Internet service providerISPCR in situ PCRIVT in vitro transcriptionJIPID Japan International Protein

Information DatabaseJPEG Joint Photographic Experts Group

(file format)K Michaelis constantkb kilobasekbps kilobits per secondKd dissociation constantkDa kilodaltonKEGG Kyoto Encyclopedia of Genes and

GenomesKHz kilohertzKLH keyhole limpet hemocyaninKmr kanamycin resistantL levorotatoryLAMP lysosome-associated proteinLAN local area networkLB Luria-Bertani (medium)LC liquid chromatographyLCL lymphoblastoid cell linesLCM laser capture microdissectionLCV lymphocryptovirus


A.1A.5


LEC ligand-exchange chromatographyLIF leukemia inhibitory factor;

laser-induced fluorescence (detector)LMPCR ligation-mediated polymerase

chain reactionLPA linear polyacrylamideLRSC lissamine rhodamineLTR long terminal repeatLumigen-PPD 4-methoxy-4-(3-phosphate

phenyl)-spiro-[1,2-dioxetane-3,2(-adamantane)], disodium salt

LysoPC lysophosphatidylcholineµµF microfaradM relative molecular weightmA milliampereMAA Maackia amurensis agglutininMAb, mAb monoclonal antibodyMAB maleic acid (buffer)MALDI matrix-assisted laser

desorption/ionization (mass spectrometry)MALDI-TOF matrix-assisted laser

desorption/ionization time-of-flight (massspectroscopy)

Man D-mannoseMAP mitogen-activated protein; multiple

antigenic peptideMb megabase, megabyteMbp megabase pairMBP maltose-binding proteinMbps megabits per secondMBS m-maleimidobenzyl-N-

hyderoxysuccinimide esterMBTH 3-methyl-2-benzothiazolinone

hydrazone hydrochlorideMCA methyl celluloacetate etherMCAC metal-chelate affinity

chromatographyMCS multiple cloning siteMDCK Madin-Darby canine kidney (cells)MDM multiply deficient medium2-ME 2-mercaptoethanolMEF mouse embryo fibroblastsMEM minimal essential mediumMEMPFA MOPS/sodium chloride/

magnesium sulfate/paraformaldehyde(buffer)

MES 2-(N-morpholino)ethanesulfonic acidMHz megahertzMIPS Martinsried Institute for Protein

SequencesmiRNA microRNAMls minor lymphocyte stimulating

determinantααMM α-methyl-D-mannosideMMC mixed-mode chromatographymmCIF macromolecular crystallographic

information file

MMDM Molecular Modeling Database ofNCBI

MMLV Moloney murine leukemia virusMMT monomethoxytritylMMTV mouse mammary tumor virusmmu millimass unit or one thousandth of a

DaltonMNase micrococcal nucleaseMOI multiplicity of infectionMolMovDB Database of Macromolecular

MovementMoMuLV Moloney murine leukemia virusMOPS 3-(N-morpholino)propane sulfonic

acidmp melting pointMPA mycophenolic acidMPC magnetic plate chamberMPSS Massive Parallel Signature

SequencingmRNA messenger ribonucleic acidMS mass spectroscopyMSCV murine stem cell virusMS/MS tandem mass spectrometryMSX methionine sulfoximineMtv mammary tumor virus designationMTX methotrexateMUG 4-methylumbelliferyl-β-D-galactosideMUP methylumbelliferyl phosphateMVA Modified vaccinia virus AnkaraMWCO molecular weight cutoffNA not applicableNAD nicotinamide adenine dinucleotideNa-DOC sodium deoxycholateNBF neutral buffered formalinNBRF National Biomedical Research

FoundationNBT nitroblue tetrazoliumNCAM neuronal cell adhesion moleculeNCBI National Center for Biotechnology

InformationNCI National Cancer InstituteNCS newborn calf serumND not determinedNDV Newcastle Disease VirusNGF nerve growth factorNLM National Library of Medicineneo neomycin gene (selectable marker)NEPHGE nonequilibrium pH gradient

electrophoresisNeu5Ac N-acetyl-D-neuraminic acidNeu5Gc N-glycolyl-D-neuraminic acidNHS N-hydroxysuccinimideNICHD National Institute of Child Health

and Human DevelopmentNIH National Institutes of HealthNK natural killer (cells)NLM National Library of Medicine

CommonAbbreviations

A.1A.6


NMR nuclear magnetic resonanceNP-40 Nonidet P-40 (detergent)NPC normal-phase chromatographyNPP nitrophenyl phosphatenr nonredundantnt nucleotideNTA nitrilotriacetic acidNTA-SAM nitrilotriacetic acid

self-assembled monolayerNTP nucleoside triphosphateOCT optimal cutting temperature (medium)o.d. outer diameterOD260 optical density at 260 nmOGT O-GlcNAc transferaseoligo oligonucleotide, a short,

single-stranded DNA or RNA.oligo(dT) oligodeoxythymidylic acidOMIM Online Mendelian Inheritance in

ManONPG o-nitrophenyl-β-D-galactosidaseORC origin recognition complexORF open reading frameori origin of replicationPAC P1-derived artificial chromosome;

phenoxyacetylPAD pulsed amperometric detectionPAGE polyacrylamide gel electrophoresisPAH polyaromatic hydrocarbonsPAP peroxidase-anti-peroxidase (reaction)par partition loci on plasmid DNAPB phosphate bufferPBMC peripheral blood mononuclear cellsPBS phosphate-buffered salinePCD programmed cell deathPCMB parachloromercuric benzoatePCR polymerase chain reactionPDB Protein Data BankPDD Protein Disease DatabasePDMP 1-phenyl-2-decanoylamino-3-

morpholino-1-propanolPE phycoerythrinPEEK polyethylether ketonePEG polyethylene glycolPEGA polyethylene glycol polyacrylamidePEI polyethyleniminePEO polyethylene oxidePFA paraformaldehydePFGE pulsed-field gel electrophoresispfu plaque-forming unitsPG proteoglycanPGK phosphoglycerate kinasepI isoelectric pointPI phosphatidylinositol; propidium iodidePI-PLC phosphatidylinositol-specific

phospholipase CPIR Protein Information ResourcePITC phenylisothiocyanate

PIPES piperazine-N,N′-bis(2-ethanesulfonic acid)

PITC phenylisothiocyanatePKC protein kinase CPLAP placental alkaline phosphatasePMF peptide mass fingerprintPMS pregnant mare’s serumPMSF phenylmethylsulfonyl fluoridePMT photomultiplier tubePNA peanut agglutininPNGase-F Peptide:N-Glycosidase FPNP purine nucleoside kinasepNP p-nitrophenolPNPP p-nitrophenyl phosphatepoly(A) polyadenylic acid or polyadenylatepoly(A)+ polyadenylated (mRNA)poly(A)− nonpolyadenylated (mRNA)poly(dA-dT) poly(deoxyadenylic

acid-deoxythymidylic acid)poly(U) polyuridylic acid or polyuridylatePP1 protein phosphatase 1PP2A protein phosphatase 2APP2B protein phosphatase 2BPPase thermostable pyrophosphataseppm parts per millionPPO 2,5-diphenyloxazolePR prosthetic-group removingPRF Protein Research FoundationPRINS primed in situ (labeling)Pristane 2,6,10,14-tetramethylpentadecaneProTherm Thermodynamic Database for

Proteins and MutantsPS copoly(styrene-1%-divinylbenzene)PSF point-spread functionPSI-BLAST Position-Specific Iterated

BLASTPth 3-phenyl-2-thiohydrantoinPTP protein tyrosine phosphatasePu purinePVA polyvinyl alcoholPVC polyvinyl chloridePVDF polyvinylidene difluoridePVP polyvinylpyrrolidonePy pyrimidinePyAOP 7-azabenzotriazol-1-yl-

oxytris(pyrrolidino)phosphoniumhexafluorophosphate

PyBOP benzotriazolyl-N-oxy-tris(pyrrolidino)phosphoniumhexafluorophosphate

rA riboadenylateRACE rapid amplification of cDNA endsRAS Ribi adjuvant systemRBC red blood cellRBE relative biological effectivenessRBS ribosome-binding siteRCA I Ricinus communis agglutinin


A.1A.7


RCF relative centrifugal forceRDA representational difference analysisRE restriction endonucleaseRed-gal 6-chloro-3-indoyl-β-D-

galactopyranosideRF replicative formRFLP restriction-fragment-length

polymorphismsRIA radioimmunoassayRIPA RadioImmunoPrecipitation AssayRMDD restriction-mediated differential

displayRNA ribonucleic acidRNAi RNA interferenceRNase ribonucleaseRP reversed phase (HPLC)RRE Rev-responsive elementrRNA ribosomal ribonucleic acidRT reverse transcriptaseRT-PCR reverse transcription/polymerase

chain reactionRU resonance unitRXR retinoid X receptorSAGE serial analysis of gene expressionSAM S-adenosylmethionineSarkosyl N-lauroylsarcosineSAX strong anion exchangeSBH sequencing by hybridizationSCOP Structural Classification of ProteinsSCX strong cation exchangeSD standard deviationSDS sodium dodecyl sulfateSE size exclusion (chromatography)SEAP secreted alkaline phosphataseSEC size-exclusion chromatographySED standard enzyme diluentSELDI surface-enhanced laser

desorption/ionizationSFFV spleen focus-forming virusSHMT serine hydroxymethyl synthetaseShrAP shrimp alkaline phosphataseSia sialic acidsiRNA short interfering RNASM suspension mediumSNA Sambucus nigra agglutininSNP single-nucleotide polymorphismSPF specific pathogen freeSPPS solid-phase peptide synthesisSPR surface plasmon resonanceSPRI solid-phase reversible immobilizationSPW surface plasma or plasmon waveSRBC sheep red blood cellsSREBP steroid response element binding

proteinss single strandedSSB single-stranded DNA-binding proteinSSC sodium chloride/sodium citrate (buffer);

side (light) scatter (in flow cytometry)

SSCP single-stranded conformationpolymorphism

SSAV simian sarcoma-associated virussss sheared salmon spermSTBS suspension Tris-buffered salineSTO SIM mouse embryo fibroblasts

resistant to thioguanine and oubainSTS sequence tagged siteSTZ streptozotocinSv Sievert (unit for radiation dosage)T thymine or thymidine; one-letter code for

threonineTAE Tris/acetate (buffer)Taq Thermus aquaticus DNA (polymerase)TAU Triton/acetic acid/ureaTBE Tris/borate (buffer)TBP TATA box-binding proteinTBS Tris-buffered salineTBT TATA-binding proteinTBTU O-benzotriazol-1-yl-N,N,N′,N′-

tetramethyluronium tetrafluoroborateTCA trichloracetic acidTCEP tris(2-carboxyethyl)phosphineTCP Transmission Control ProtocolTCR T cell receptorTDM trehalose dimycolateTdT terminal deoxynucleotidyl transferaseTE Tris/EDTA (buffer)TEA triethanolamine acetateTEAE triethylaminoethylTEMED N,N,N′,N′-

tetramethylethylenediamineTEN NaCl in TE bufferTES N-tris(hydroxymethyl)methyl-2-

aminoethanesulfonic acidTFA trifluoroacetic acidTFFH tetramethylfluoroformamidinium

hexafluorophosphateTFMSA trifluoromethanesulfonic acidTGN trans-Golgi networkTHF tetrahydrofuranTIFF tagged-image file formatTIR total internal reflectionTK thymidine kinaseTLC thin-layer chromatographyTLD thermoluminescent dosimeterTm melting (or midpoint) temperature;

thermal denaturationTMAC tetramethylammonium chlorideTMB 3,3′,5,5′-tetramethylbenzidineTMP trimethylphosphate; thymidine

monophosphateTMV Tobacco Mosaic VirusTONPG orthonitrophenyl-β-D-

thiogalactosideTPCK N-p-tosyl-L-phenylalanine

chloromethyl ketoneTPF tiling path format

CommonAbbreviations

A.1A.8


Tris tris(hydroxymethyl)aminomethaneTRITC tetramethylrhodamine

isothiocyanatetRNA transfer ribonucleic acidTS thymidylate synthetaseTSA Tris/saline/azide (buffer)TTP thymidine 5′-triphosphateTUNEL TdT-mediated dUTP biotin

nick-end labelingU unit; uracil or uridineUAS upstream activating sequenceUDG uracil DNA glycosylaseUDP uridine 5′-diphosphateUDP-Gal uridine diphospho-D-galactoseUF ultrafiltrationUMP uridine 5′-monophosphateUPHS U.S. Public Health ServiceURL uniform resource locatorUSDA United States Department of

AgricultureUTP uridine 5′-triphosphateUTR untranslated leader regionUV ultravioletUWGCG University of Wisconsin Genetics

Computer GroupVAF viral-antibody freeVAST Vector Alignment Search ToolVent Thermococcus litoralis DNA

(polymerase)

VRC vanadyl-ribonucleoside complexV0 void volumevol/vol; v/v volume/volumeVSG variant surface glycoproteinVSV vesicular stomatitis virusWAIS Wide Area Information ServiceWAX weak anion exchangeWCX weak cation exchangeWGA wheat germ agglutininWR Western Reserve strain (vaccinia)WT wild-typewt/vol; w/v weight/volumeWWW World Wide WebXBE Rex-binding elementXgal 5-bromo-4-chloro-3-indolyl-β-D-

galactosideXGPRT xanthine-guanine phosphoribosyl

transferaseXyl xyloseXyl-A 9-β-D-xylofuranosyl adenineYAC yeast artificial chromosomeYCp yeast centromeric plasmidYEp yeast episomal plasmidYIp yeast integrating plasmidYNB-AA/AS yeast nitrogen base without

amino acids or ammonium sulfateYPD yeast/peptone/dextrose (medium)YRp yeast replicating plasmid

APPENDIX 1BUseful Measurements and Data

Table A.1B.1 Conversion Factors

Molecular weight (ave.) of DNA base pair: 649 Da 1 kb DNA: 333 amino acids of coding capacityMolecular weight (ave.) of amino acid: 110 Da ≈ 36,000 Da1 µg/ml DNA: 3.08 µM phosphate 6.5 × 105 Da of double-stranded DNA (sodium salt)1 µg/ml of 1 kb DNA: 3.08 nM 5′ ends 3.3 × 105 Da of single-stranded DNA (sodium salt)1 µmol pBR322 (4363 bp): 2.83 g 3.4 × 105 Da of single-stranded RNA (sodium salt)1 pmol linear pBR322 5′ ends: 1.4 µg1 A260 double-stranded DNA: 50 µg/ml 10 kDa protein ≈ 91 amino acids1 A260 single-stranded DNA: 37 µg/ml ≈ 273 nucleotides

Supplement 44

Current Protocols in Molecular Biology (1997) A.1B.1Copyright © 1997 by John Wiley & Sons, Inc.

Table A.1B.2 Genome Size of Various Organismsa

Organism Base pairs/haploid genome Organism Base pairs/

haploid genome

SV40 5,243 Drosophila melanogaster 1.4 × 108

ΦX174 5,386 Gallus domesticus (chicken) 1.2 × 109

Adenovirus 2 35,937 Mus musculus (mouse) 2.7 × 109

Lambda 48,502 Rattus norvigeticus (rat) 3.0 × 109

Escherichia coli 4.7 × 106 Xenopus laevis 3.1 × 109

Saccharomyces cerevisiae 1.5 × 107 Homo sapiens 3.3 × 109

Dictyostelium discoideum 5.4 × 107 Zea mays 3.9 × 109

Arabidopsis thaliana 7.0 × 107 Nicotiana tabacum 4.8 × 109

Caenorhabditis elegans 8.0 × 107

aGenome size determined either by direct sequence analysis (viruses), electrophoretic analysis (E. coli, S. cerevisiae), ora combination of DNA content per cell and hybridization kinetics. Some data are from Gene Expression 2 by Lewin, 1980.

SMALL MOLECULES

BIG MOLECULES

22% (ww) proteins, nucleicacids, polysaccharides

70% (ww) water 3% (ww) sugars (monomers) 2% (ww) lipids0.4% (ww) amino acids (monomers)0.4% (ww) nucleotides (monomers)

140 mM 5 mM30 mM Mg2+ 1-2 mM

Ca2+ 1-2 mM 2.5 -5 mM(although <10–7 M is free)

Cl– 4 mM 110 mM

INORGANIC IONS (ca. 1% w/w)

pH = 7.4

DIAMETER

Prokaryotes:<1-10 µm

Eukaryotes:10-120 µm

Na+ 5 -15 mM 145 mM

K+

Figure A.1B.1 A physical chemist’s view of the cell. The data in this figurewere assembled from The Molecular Biology of the Cell by Alberts, 1994,and represent the approximate concentrations of a variety of intracellularcomponents.

A.1B.1


APPENDIX 1C Characteristics of Amino Acids

PHYSICAL PROPERTIES

The physical properties of the amino acids determine the structure and function of theproteins in which they are found. Some useful details and relevant physical characteristicsof the amino acids can be found in Table A.1C.1. A detailed view of the chemical structuresof the amino acids, and an explanation of the role these structures play in enzymes, canbe found in Figure A.1C.2. The three-dimensional structure of proteins is largely deter-mined by the packing of their hydrophobic cores; the properties of amino acids that governthis packing are their relative hydrophobicities, which are presented in Figure A.1C.3,and their shapes and volumes, which can be assessed by referring to the space-fillingmodels shown in Figure A.1C.4. While these figures can be useful in rationalizing aminoacid functionality, it is also important to consider how natural selection views theinterchangeability of amino acids, as diagrammed in Figure A.1C.5 (and Table A.1C.1).

Post-translational modifications will change the mass of a protein or peptide; values forsome common mass changes are listed in Table A.1C.3. Mass changes due to somepost-translational modifications are found in Table A.1C.4.

CODON USAGE (see Table A.1C.2)

While the amino acid sequence of a protein is selected for in part because of the physicalproperties of the amino acids themselves, a second, more subtle selection may also operateat the level of the genetic code to determine the sequence of both protein and gene. Thegenetic code is degenerate. Any of several codons can represent a single amino acid (upto six, in the cases of Arg, Leu, and Ser). However, the frequency with which suchsynonymous codons are used is not equivalent. Considerations of the bias in codon usagemay be relevant to design of synthetic genes (UNIT 8.2B), strategies for overexpression offoreign proteins, particularly in E. coli (see UNIT 16.1), and minimizing degeneracy ofoligonucleotide probes and primers (UNIT 6.4 and UNIT 15.1).

The reasons for deviation from random usage seem to differ from organism to organism.For E. coli and other microorganisms, it is thought that the codons used more frequentlycorrespond to abundant tRNAs, while the underrepresented codons are those associatedwith less abundant tRNAs. Since this bias seems particularly strong for genes encodinghighly expressed proteins, it is thought to be related to maximizing translation efficiency.In higher organisms, the bias in codon usage may be more closely associated withselection pressures acting at the level of DNA. Mammalian genomes, in particular, showquite significant reductions in the frequency of the dinucleotide CpG, which is a site formethylation. In mammalian genes, codons containing this sequence can be quite stronglyunderrepresented.

It is worth emphasizing that the bias against particular codons is not absolute. While theremay be strong trends within a particular organism, individual genes (particularly thoseexpressed at low levels) may deviate substantially (see Sharp et al., 1988). Interestingly,codon usage is quite similar within the broad groups presented (see Wada et al., 1990, fora comparison of mammals).

Contributed by Andrew Ellington and J. Michael CherryCurrent Protocols in Molecular Biology (1997) A.1C.1-A.1C.12Copyright © 1997 by John Wiley & Sons, Inc.

Supplement 44

A.1C.1

Characteristics ofAmino Acids

Table A.1C.1 Physical Characteristics of the Amino Acids

Amino acid 3-lettercode

1-lettercode

Mol. wt.(g/mol)

Accessiblesurface areaa

Hydro-phobicityb

Relativemutabilityc

Surfaceprobabilityd

Alanine Ala A 89.1 115 −0.40 100 62Arginine Arg R 174.2 225 −0.59 65 99Asparagine Asn N 132.1 160 −0.92 134 88Aspartate Asp D 133.1 150 −1.31 106 85Cysteine Cys C 121.2 135 0.17 20 55Glutamate Glu E 147.1 190 −1.22 102 82Glutamine Gln Q 146.2 180 −0.91 93 93Glycine Gly G 75.1 75 −0.67 49 64Histidine His H 155.2 195 −0.64 66 83Isoleucine Ile I 131.2 175 1.25 96 40Leucine Leu L 131.2 170 1.22 40 55Lysine Lys K 146.2 200 −0.67 56 97Methionine Met M 149.2 185 1.02 94 60Phenylalanine Phe F 165.2 210 1.92 41 50Proline Pro P 115.1 145 −0.49 56 82Serine Ser S 105.1 115 −0.55 120 78Threonine Thr T 119.1 140 −0.28 97 77Tryptophan Trp W 204.2 255 0.50 18 73Tyrosine Tyr Y 181.2 230 1.67 41 85Valine Val V 117.1 155 0.91 74 46aAccessible surface area is in Å2 and is for the amino acid as part of a polypeptide backbone (Chothia, 1976).bHydrophobicity is in arbitrary units and is based on the OMH scale of Sweet and Eisenberg (1983), which emphasizesthe ability of amino acids to replace one another during the course of evolution.cRelative mutability is also in arbitrary units (with alanine set to 100) and represents the probability that an amino acidwill mutate within a given time. Thus, as two closely related proteins diverge, a given tryptophan residue is only 18% aslikely as a given alanine residue to mutate (Dayhoff et al., 1978).dSurface probability is the likelihood that 5% or more of the surface area of an amino acid will be exposed to the solutionsurrounding a protein (Chothia, 1976). Thus, while some portion of almost all the arginines will help make up the surfaceof a protein, less than half of the valines will be exposed to solution. To understand in more detail how amino acids areburied, see Rose et al. (1985); for example, although tyrosine is often found exposed to the surface of a protein, a substantialproportion of its surface area is typically buried.

Figure A.1C.1 The genetic code.Names of amino acids and chaintermination codons are on theperiphery of the circle. The first baseof the codon is identified in thecenter ring; the second base of thecodon is in the middle ring; and thethird base(s) of the codon is in theouter ring of the circle.

Gly

Ala

Ser

Tyr

CysUGATrp

Leu

Pro

HisGln

ArgIIe

Thr

Asn

Lys

Ser

Arg

Val

Asp

Glu P

he

Leu

ochre

amberC

A

G

T

C

AGT

C

A

G

T

C

AG T

GACT

GA

CT

GACTG

AC

TG

AC T G A C T G A C T

GA CTG

ACTGACTGA

CT

GA

TC

AG

CTGACTGAT CGA

CT

TG

A C

Met


A.1C.2


Tab

le A

.1C

.2

Per

cent

age

of C

odon

Syn

onom

ous

Usa

ge a

nd F

requ

ency

of C

odon

Occ

urre

nce

in V

ario

us O

rgan

ism

s (s

ee d

escr

iptio

n be

low

)

AA

Cod

onM

amm

alO

ther

ver

t.D

icot

Mon

ocot

Inve

rteb

rate

Yea

stC

hlor

opla

stY

east

mit

o.G

ram

neg

.G

ram

pos

.%

freq

.%

freq

.%

freq

.%

freq

.%

freq

.%

freq

.%

freq

.%

freq

.%

freq

.%

freq

.

Gly

GG

G22

.816

.620

.714

.811

.69.

425

.020

.58.

46.

09.

05.

215

.313

.86.

03.

812

.39.

413

.79.

2G

lyG

GA

25.5

18.5

29.2

20.9

37.9

30.6

21.2

17.4

32.4

23.3

15.0

8.5

38.1

34.4

21.0

12.8

9.2

7.0

30.5

20.5

Gly

GG

U17

.612

.821

.315

.235

.628

.815

.212

.421

.415

.661

.034

.935

.632

.268

.040

.932

.925

.127

.218

.2G

lyG

GC

34.1

24.7

28.9

20.7

14.9

12.1

38.6

31.6

37.8

25.7

15.0

8.9

11.0

9.9

4.0

2.2

45.6

34.9

28.5

19.1

Glu

GA

G60

.140

.455

.942

.348

.929

.372

.531

.966

.038

.426

.017

.023

.714

.420

.07.

234

.521

.226

.619

.3G

luG

AA

39.9

26.8

44.1

33.4

51.1

30.6

27.5

12.1

34.0

20.7

74.0

49.4

76.3

46.5

80.0

28.9

65.5

40.2

73.4

53.2

Asp

GA

U42

.620

.950

.926

.260

.126

.429

.710

.253

.025

.262

.037

.275

.428

.172

.026

.755

.230

.967

.638

.8A

spG

AC

57.4

28.1

49.1

25.3

39.9

17.5

70.3

24.1

47.0

22.4

38.0

22.6

24.6

9.2

28.0

10.4

44.8

25.1

32.4

18.6

Val

GU

G48

.129

.542

.025

.728

.619

.338

.023

.441

.622

.815

.09.

615

.29.

98.

04.

634

.223

.821

.913

.7V

alG

UA

9.9

6.1

13.1

8.0

14.0

9.4

9.5

5.8

10.6

6.1

16.0

9.7

40.6

26.4

46.0

27.7

16.0

11.1

25.8

16.1

Val

GU

U16

.410

.022

.013

.540

.026

.918

.811

.621

.112

.044

.027

.434

.322

.337

.022

.125

.918

.032

.220

.1V

alG

UC

25.6

15.7

22.9

14.0

17.4

11.7

33.7

20.8

26.8

15.3

24.0

15.1

9.9

6.4

9.0

5.2

23.9

16.6

20.0

12.5

Ala

GC

G9.

97.

08.

86.

56.

64.

825

.321

.718

.113

.98.

05.

010

.17.

75.

03.

234

.333

.321

.414

.7A

laG

CA

21.0

14.9

26.7

19.6

27.0

19.4

18.0

15.5

18.2

14.7

23.0

15.0

26.4

20.2

36.0

21.7

19.3

18.7

35.7

24.6

Ala

GC

U28

.820

.433

.224

.444

.632

.021

.818

.721

.417

.344

.028

.548

.537

.151

.030

.717

.216

.727

.619

.0A

laG

CC

40.2

28.5

31.3

23.0

21.8

15.6

34.9

30.0

42.4

33.0

25.0

16.0

15.0

11.5

8.0

4.8

29.2

28.3

15.4

10.6

Arg

AG

G21

.511

.821

.412

.025

.111

.924

.412

.714

.19.

417

.07.

39.

36.

311

.03.

23.

21.

99.

93.

8A

rgA

GA

21.0

11.5

23.6

13.2

32.6

15.4

13.0

6.8

14.6

9.8

54.0

23.6

30.1

20.2

70.0

20.3

4.3

2.5

33.1

12.6

Ser

AG

U13

.410

.013

.29.

616

.412

.87.

75.

512

.410

.414

.011

.119

.712

.815

.011

.011

.87.

014

.69.

9Se

rA

GC

24.8

18.6

25.8

18.7

17.3

13.5

22.1

15.6

22.4

18.4

9.0

7.4

10.1

6.6

5.0

3.4

28.0

16.6

20.5

13.9

Lys

AA

G62

.436

.158

.438

.057

.733

.080

.528

.870

.637

.049

.035

.328

.714

.625

.013

.629

.514

.224

.219

.8Ly

sA

AA

37.6

21.7

41.6

27.1

42.3

24.2

19.5

7.0

29.4

15.5

51.0

36.8

71.3

36.1

75.0

41.7

70.5

33.9

75.8

61.8

Asn

AA

U41

.415

.543

.817

.045

.920

.725

.97.

644

.118

.954

.031

.066

.525

.286

.066

.237

.815

.558

.231

.3A

snA

AC

58.6

22.0

56.2

21.8

54.1

24.4

74.1

21.9

55.9

24.3

46.0

25.9

33.5

12.7

14.0

10.8

62.2

25.6

41.8

22.5

Met

AU

G10

022

.610

023

.410

022

.310

020

.610

023

.210

021

.310

024

.065

.028

.710

025

.210

023

.4Il

eA

UA

13.1

5.9

16.8

7.8

18.9

9.9

13.6

5.5

15.6

7.1

20.0

12.1

24.5

18.9

35.0

15.4

7.3

4.1

18.7

12.4

Ile

AU

U33

.314

.935

.816

.746

.224

.227

.511

.133

.815

.450

.030

.751

.739

.986

.067

.842

.623

.951

.534

.2Il

eA

UC

53.6

24.0

47.4

22.0

34.9

18.2

58.9

23.8

50.6

23.5

30.0

18.4

23.8

18.4

14.0

11.2

50.1

28.0

29.8

19.8

Thr

AC

G11

.86.

611

.36.

38.

54.

721

.110

.724

.713

.812

.06.

89.

04.

86.

02.

824

.413

.420

.311

.8T

hrA

CA

26.4

14.8

30.3

17.1

29.3

16.1

18.9

9.6

21.8

12.7

26.0

15.4

30.1

16.0

46.0

20.1

11.5

6.3

47.3

27.4

Thr

AC

U23

.413

.126

.014

.735

.019

.320

.910

.618

.010

.238

.022

.540

.421

.638

.016

.718

.810

.321

.312

.4T

hrA

CC

38.5

21.6

32.4

18.3

27.2

15.0

39.0

19.8

35.5

20.2

25.0

14.5

20.5

10.9

10.0

4.4

45.3

24.9

11.0

6.4

Trp

UG

G10

014

.410

012

.510

014

.710

013

.010

011

.610

010

.210

011

.939

.06.

810

013

.310

09.

7E

ndU

GA

59.6

2.6

58.1

2.9

51.4

3.8

60.3

3.7

56.2

3.9

34.0

0.7

23.1

0.9

61.0

10.6

33.2

1.0

18.9

0.6

Cys

UG

U42

.710

.039

.78.

749

.38.

927

.05.

531

.57.

368

.07.

971

.46.

687

.06.

638

.04.

247

.83.

0C

ysU

GC

57.3

13.4

60.3

13.2

50.7

9.2

73.0

14.9

68.5

15.1

32.0

3.8

28.6

2.6

13.0

1.0

62.0

6.8

52.2

3.3

End

UA

G17

.20.

713

.80.

718

.01.

321

.31.

315

.31.

017

.00.

415

.40.

67.

00.

29.

70.

315

.60.

5E

ndU

AA

23.2

1.0

28.0

1.4

30.6

2.3

18.4

1.1

28.5

2.0

49.0

1.0

61.5

2.3

93.0

2.6

57.2

1.8

65.6

2.1

Tyr

UA

U40

.211

.541

.611

.246

.914

.425

.77.

336

.99.

450

.016

.371

.623

.282

.036

.350

.615

.270

.626

.3Ty

rU

AC

59.8

17.2

58.4

15.8

53.1

16.3

74.3

21.1

63.1

16.2

50.0

16.6

28.4

9.2

18.0

8.2

49.4

14.8

29.4

11.0

Leu

UU

G12

.211

.413

.812

.026

.123

.015

.713

.016

.813

.436

.032

.621

.519

.710

.013

.211

.611

.115

.012

.9L

euU

UA

5.4

5.0

6.5

5.6

11.1

9.8

4.6

3.8

7.0

5.6

27.0

24.1

31.5

28.8

72.0

92.3

9.4

9.0

29.5

25.4

Phe

UU

U40

.715

.344

.214

.649

.521

.330

.610

.134

.811

.353

.022

.759

.824

.060

.034

.547

.517

.168

.027

.3Ph

eU

UC

59.3

22.3

55.8

18.4

50.5

21.8

69.4

22.9

65.2

21.2

47.0

20.0

40.2

16.2

40.0

23.1

52.5

18.9

32.0

12.8

A.1C.3


Ser

UC

G5.

94.

46.

04.

35.

84.

517

.012

.019

.515

.78.

06.

57.

04.

54.

03.

015

.49.

18.

65.

8Se

rU

CA

14.2

10.6

15.4

11.2

19.7

15.3

16.0

11.3

11.4

9.9

19.0

15.3

16.1

10.5

44.0

31.5

10.7

6.4

22.2

15.0

Ser

UC

U18

.313

.819

.314

.023

.618

.315

.310

.811

.39.

732

.025

.427

.117

.625

.017

.716

.19.

623

.415

.9Se

rU

CC

23.5

17.6

20.4

14.8

17.2

13.4

21.9

15.5

23.0

18.9

18.0

14.8

20.0

13.0

7.0

4.8

18.0

10.7

10.7

7.3

Arg

CG

G18

.210

.013

.37.

55.

02.

416

.48.

613

.88.

72.

01.

06.

14.

14.

01.

29.

65.

610

.84.

1A

rgC

GA

10.7

5.8

9.4

5.3

9.5

4.5

9.0

4.7

14.1

9.3

5.0

2.1

20.1

13.5

1.0

0.2

5.6

3.3

10.5

4.0

Arg

CG

U9.

15.

013

.47.

518

.88.

911

.25.

816

.510

.917

.07.

325

.517

.213

.03.

636

.921

.419

.97.

6A

rgC

GC

19.5

10.7

18.9

10.6

9.0

4.2

26.0

13.6

27.0

17.0

4.0

1.9

8.9

6.0

1.0

0.4

40.4

23.5

15.8

6.0

Gln

CA

G73

.932

.769

.230

.239

.715

.240

.143

.465

.032

.826

.010

.326

.89.

917

.04.

470

.229

.436

.415

.3G

lnC

AA

26.1

11.6

30.8

13.5

60.3

23.1

59.9

64.8

35.0

18.2

74.0

29.7

73.2

27.1

83.0

21.9

29.8

12.5

63.6

26.7

His

CA

U38

.89.

243

.110

.053

.012

.038

.28.

141

.612

.160

.012

.571

.514

.383

.019

.550

.111

.170

.415

.8H

isC

AC

61.2

14.5

56.9

13.3

47.0

10.7

61.8

13.2

58.4

16.5

40.0

8.3

28.5

5.7

17.0

4.0

49.9

11.1

29.6

6.6

Leu

CU

G42

.840

.140

.334

.911

.09.

628

.223

.238

.130

.09.

08.

56.

25.

75.

06.

453

.751

.518

.515

.9L

euC

UA

6.8

6.4

7.2

6.3

9.8

8.6

9.8

8.1

8.6

7.0

13.0

11.8

15.4

14.1

6.0

8.0

3.3

3.1

7.4

6.4

Leu

CU

U12

.111

.314

.812

.825

.222

.213

.411

.012

.410

.311

.09.

619

.617

.95.

06.

09.

99.

521

.618

.6L

euC

UC

20.8

19.5

17.4

15.0

16.9

14.8

28.3

23.3

17.2

14.3

4.0

4.0

5.7

5.2

1.0

1.4

12.1

11.6

8.0

6.9

Pro

CC

G11

.26.

811

.06.

08.

24.

625

.720

.527

.315

.59.

04.

112

.86.

35.

02.

054

.423

.730

.310

.8Pr

oC

CA

27.3

16.5

30.4

16.7

41.9

23.6

40.9

32.6

30.1

17.9

49.0

21.9

23.3

11.4

39.0

15.6

17.1

7.4

28.9

10.3

Pro

CC

U28

.817

.428

.615

.734

.119

.215

.012

.014

.68.

929

.012

.743

.321

.148

.019

.315

.26.

633

.211

.9Pr

oC

CC

32.7

19.7

30.0

16.4

15.9

8.9

18.3

14.6

28.0

16.1

13.0

5.7

20.6

10.0

8.0

3.4

13.3

5.8

7.6

2.7

AA

Cod

onM

amm

alO

ther

ver

t.D

icot

Mon

ocot

Inve

rteb

rate

Yea

stC

hlor

opla

stY

east

mit

o.G

ram

neg

.G

ram

pos

.%

freq

.%

freq

.%

freq

.%

freq

.%

freq

.%

freq

.%

freq

.%

freq

.%

freq

.%

freq

.

A d

iscu

ssio

n of

cod

on r

ando

mne

ss a

nd t

he r

elev

ance

of

this

phe

nom

enon

in

mol

ecul

arbi

olog

y ex

perim

enta

tion

can

be fo

und

on p

. A.1

.6. (

% C

olu

mn

) P

erce

ntag

e of

syn

onym

ous

codo

n us

age.

The

rel

ativ

e pe

rcen

tage

of

each

mem

ber

of t

he s

et o

f co

dons

tha

t sp

ecify

apa

rtic

ular

am

ino

acid

. Fou

r se

ts t

hat

are

not

cont

iguo

us in

thi

s ta

ble

are

Arg

, S

er,

Leu,

and

chai

n te

rmin

ator

. (F

req

. Co

lum

n)

Freq

uenc

ies

are

expr

esse

d as

occ

urre

nces

per

thou

sand

codo

ns fo

r the

spe

cifie

d or

gani

sms.

The

Gen

Ban

k nu

clei

c ac

id d

atab

ase

as o

f Apr

il 23

, 199

0,w

as u

sed

as t

he s

ourc

e of

gen

e-co

ding

reg

ions

. Thi

s co

rres

pond

s to

Gen

bank

Rel

ease

63

with

the

add

ition

of

thre

e w

eekl

y up

date

s. T

he fe

atur

e ta

bles

wer

e ut

ilize

d to

aut

omat

ical

lyex

trac

t pe

ptid

e-co

ding

reg

ions

fro

m t

he d

atab

ase.

Thi

s au

tom

ated

pro

cess

dis

card

ed s

e-qu

ence

s th

at w

ere

less

than

ten

codo

ns in

leng

th a

nd c

onta

ined

mor

e th

an o

ne c

hain

-ter

mi-

nat

ion

cod

on.

Th

e ex

tra

cte

d co

din

g s

equ

ence

s w

ere

the

n ex

am

ine

d to

rem

ove

all

psue

doge

nes,

non

nucl

ear

(exc

ept

for

the

chlo

ropl

ast

and

mito

chon

dria

l ca

tego

ries)

, vi

ral,

rear

rang

ed,

and

mut

ant

sequ

ence

s. T

he r

emai

ning

col

lect

ion

of e

xtra

cted

seq

uenc

es w

ased

ited

to r

emov

e du

plic

ate

entr

ies;

mos

t si

gnifi

cant

ly,

only

one

exa

mpl

e of

eac

h cl

ass

ofim

mun

oglo

bulin

gen

e w

as a

llow

ed p

er s

peci

es. T

he c

odon

freq

uenc

ies

wer

e ta

bula

ted

with

the

aid

of t

he C

odon

Freq

uenc

y pr

ogra

m f

rom

the

UW

GC

G p

acka

ge (

Gen

etic

s C

ompu

ter

Gro

up, M

adis

on, W

I 537

11).

The

tota

l num

ber

of c

odon

s us

ed fo

r ea

ch o

rgan

ism

cat

egor

y an

d th

e so

urce

of t

he c

odon

sar

e lis

ted

belo

w e

xpre

ssed

as

a pe

rcen

tage

of t

he to

tal:

Mam

mal

: Tot

al o

f 1,2

37,0

27 c

odon

sfr

om c

ow ( B

os ta

urus

; 6.4

3%),

ham

ster

(Cric

etul

us s

p. a

nd M

esoc

ricet

us s

p.; 1

.48%

), h

uman

(Hom

o sa

pien

s; 4

8.36

%),

mac

aque

(M

acac

a sp

.; 0.

38%

), m

ouse

(M

us s

p.; 1

9.52

%),

rab

bit

( Ory

ctol

agus

sp.

; 4.

06%

), r

at (

Rat

tus

sp.;

19.1

7%),

and

she

ep (

Ovi

s sp

.; 0.

59%

). O

ther

vert

ebra

te: T

otal

of 1

59, 9

94 c

odon

s fr

om c

hick

en (G

allu

s sp

.; 72

.24%

) an

d X

enop

us la

evis

(27.

76%

). D

ico

t: T

otal

of

71,

408

codo

ns f

rom

Ara

bido

psis

tha

liana

(15

.21%

), p

ea (

Pis

umsa

tivum

; 13

.77%

), P

etun

ia s

p. (

6.61

%),

lim

a be

an (

Pha

seol

us v

ulga

ris;

11.2

4%),

pot

ato

( Sol

anum

tub

eros

um; 7

.89%

), t

obac

co (

Nic

otia

na t

abac

um; 9

.54%

), t

omat

o (L

ycop

ersi

con

escu

lent

um; 1

1.63

%),

and

soy

bean

(Gly

cine

max

; 24.

10%

). M

on

oco

t: T

otal

of 4

5, 6

22 c

odon

sfr

om b

arle

y ( H

orde

um v

ulga

re;

21.3

5%),

cor

n (Z

ea m

ays

47.7

0%),

ric

e (O

ryza

sat

iva;

11.6

0%) a

nd w

heat

(Trit

icum

aes

tivum

; 19.

35%

). In

vert

ebra

te: T

otal

of 1

51, 7

94 c

odon

s fr

omC

aeno

rhab

ditis

ele

gans

(9.

11%

), s

ea u

rchi

n (S

tron

gylo

cent

rotu

s pu

rpur

atus

; 6.

01%

), a

ndfr

uit

fly (

Dro

soph

ila m

elan

ogas

ter;

84.

88%

). Ye

ast:

Tot

al o

f 21

6, 3

75 c

odon

s fr

om S

ac-

char

omyc

es c

erev

isia

e. C

hlo

rop

last

: To

tal

of 6

, 86

6 co

dons

fro

m Z

ea m

ays

chlo

ropl

ast

(52.

18%

) and

Nic

otia

na ta

bacu

m c

hlor

opla

st (4

7.82

%).

Yea

st m

ito

cho

nd

rio

n: T

otal

of 4

, 986

codo

ns f

rom

Sac

char

omyc

es c

erev

isia

e m

itoch

ondr

ia (

see

note

bel

ow).

Gra

m n

egat

ive

bac

teri

a: T

otal

of

263,

904

cod

ons

from

Esc

heric

hia

coli

(70.

40%

), K

lebs

iella

pne

umon

iae

( 3.9

6%),

Nei

sser

ia g

onor

rhea

e (1

.75%

), P

seud

omon

as s

p. (

10.9

7%),

Rhi

zobi

um m

elilo

ti(2

.57%

), a

nd S

alm

onel

la t

yphi

mur

ium

(10

.35%

). G

ram

po

siti

ve b

acte

ria:

Tot

al o

f 38

,807

codo

ns fr

om B

acill

us s

ubtil

is (

73.4

5%)

and

Sta

phyl

ococ

cus

aure

us (

26.5

5%).

NO

TE

: T

he g

enet

ic c

ode

is u

nive

rsal

with

the

exc

eptio

n of

mito

chon

dria

l D

NA

. In

yea

stm

itoch

ondr

ia th

e A

UA

and

UG

A c

odon

s th

at n

orm

ally

spe

cify

isol

euci

ne a

nd c

hain

term

inat

ion

are

used

for

met

hion

ine

and

tryp

toph

an,

resp

ectiv

ely.

The

se e

xcep

tions

are

use

d w

hen

calc

ulat

ing

the

yeas

t mito

chon

drio

n pe

rcen

tage

of s

ynon

ymou

s co

don

usag

e.

A.1C.4


+H3N COO–

A

Amino acids with dissociable protons

acidic10.5

OH

+H3N COO–

H

3.9COOH

+H3N COO–

H

8.4SH

+H3N COO–

H

13.7OH

+H3N COO–

H

COOH

+H3N COO–

H

4.1

pKa3 4 5 6 7 8 9 10 11 12 13 14

serinetyrosinecysteineglutamateaspartate

6.0 10.5

12.5

+H3N COO– +H3N COO–

argininelysinehistidine

basic

Other amino acids with polar side chains

+H3N COO–

NH2

H

O

asparagine

+H3N COO–

NH2

O

H

glutamine

+H3N COO–

HH

OH

H3C

threonine

NH

NH2+

NH2

HHH

NH3+H

N

+

N H

Fig. A.1C.2


A.1C.5


Nonpolaramino acids

+H3N COO–

H

+H3N COO–

H

glycine

tryptophan

N COO–

H

proline

+H3N COO–

H

alanine

H

HH +

H3C

+H3N COO–

H

valine

+H3N COO–

H

methionine

HN

CH3

H3C

S

CH3

+H3N COO–

H

leucine

+H3N COO–

H

isoleucine

+H3N COO–

H

phenylalanine

CH3CH3H

CH3

H3C

B


Figure A.1C.2 Line drawings of the amino acids. Theamino acids are roughly divided into three groups: aminoacids with dissociable protons (A), other amino acids withpolar side chains (A), and nonpolar amino acids (B). Thesegroupings are designed to facilitate an understanding ofenzymology and the thermodynamics of protein folding.

In this representation, hydrogens are omitted except inshowing ionization or stereochemistry. In the case of ar-ginine the delocalized positive charge is indicated bydashed double bonds. At stereocenters, bold lines indicatea group is coming out of the page toward the viewer, whilehashed lines indicate that the group goes into the page awayfrom the viewer.

Amino acids with dissociable protons are generally inti-mately involved in the chemistry of enzymes. Acidic andbasic groups can form salt bridges to substrates or to eachother. They can also act as proton donors/acceptors inmechanisms that rely on acid/base catalysis. The polar sidechains of some of these amino acids (notably cysteine,serine, and histidine) can act as nucleophiles. The pKa

values for the free amino acids are shown, but these values

can markedly change when these groups are buried inproteins. The pKas of the α-amino groups range from 8.7 to10.7, while the pKas of the α-carboxylates range from 1.8 to2.4.

Amino acids with polar side chains (A) can form hydrogenbonds to substrates or to each other. Cysteine, serine, andtyrosine could also be included in this group, since theionized forms of these amino acids do not generally performstructural roles in proteins. In general, these amino acids(and the amino acids with dissociable protons) will be foundon the surfaces of proteins. Cysteine is an exception, sinceit is slightly hydrophobic and can often be buried as adisulfide bond.

The nonpolar amino acids (B) are often found in the interiorsof proteins or in hydrophobic substrate-binding pockets.They interact with one another like jigsaw pieces, formingtight-fitting associations that have a density similar to that ofan amino acid crystal. Proline is buried less frequently thanmight be expected because of its predominance in turns,which are often found on the periphery of a protein.

A.1C.6


OMH (arbitrary units)

Frömmel (kcal/mol)

F Y I L M V W C T A P S R H G K Q N D E

Amino acid

Hyd

roph

obic

ity

3

2

1

0

–1

–2

–3

Figure A.1C.3 Amino acid hydrophobicity. The hydrophobicity of an amino acid is the degree towhich it prefers a nonpolar medium, such as ethanol or the interior of a protein, to a polar medium,such as water. In this graph, the more hydrophobic amino acids “sink” below zero, while the morehydrophilic amino acids “float” above the surface.

Two scales are used. The Frömmel scale (Frömmel, 1984) represents the free energy of transferfrom a hydrophobic medium to water. This value is an intrinsic property of an amino acid, separatefrom its role in a protein. In contrast, the OMH scale (Sweet and Eisenberg, 1983) is a measure ofhow likely a given amino acid will be replaced by a different hydrophobic or “buried” amino acid ina protein. In effect, this scale is how evolution views the hydrophobicity of an amino acid.

The distinction between physical and evolutionary properties is important. For example, whilearginine is definitely a charged, polar amino acid (Sambrook et al., 1989), it can substitute morefreely for nonpolar amino acids in the interior of a protein than glutamate (also a charged, polaramino acid) because of its long aliphatic side chain.


A.1C.7


C N

S

H

O

glycine alanine serine cysteine threonine proline

aspartate asparagine leucine isoleucine glutaminevaline

methionine histidine lysine phenylalanineglutamate

arginine tyrosine tryptophan

Figure A.1C.4 Space-filling representations of the amino acids. The amino acids are arranged in order of size. Theconformations shown maximize the two-dimensional area but are not necessarily the most stable geometries.


A.1C.8


W

H

Q

R K

N

SA

P

G

V I

T

C

E D

L

F YM

Figure A.1C.5 Mutational pathways for amino acids. In this diagram, amino acids are parsed intosets based on their ability to replace one another during the evolution of closely related proteins.Dark arrows show the most frequent mutational events for each of the twenty amino acids. Forexample, tryptophan most frequently mutates to arginine, while arginine and lysine most frequentlyreplace one another. Dotted arrows represent the most frequent replacements between sets ofotherwise mutationally related amino acids. Thus, while lysine mutates most frequently to argininewithin the [arginine, lysine, tryptophan] set, the most likely event that will occur outside of this setis mutation to asparagine.


A.1C.9


Table A.1C.3 Compositions and Masses of the Twenty Commonly Occurring Amino Acid Residuesa,b

Name Composition Monoisotopic mass

Average mass

pKa of ionizingside chainc

Occurrence inproteins (%)

Alanine (Ala, A) C3H5NO 71.03711 71.0788 — 8.3

Arginine (Arg, R) C6H12N4O 156.10111 156.1876 ~11.5–12.5 (12) 5.7

Asparagine (Asn, N) C4H6N2O2 114.04293 114.1039 — 4.4

Aspartic acid (Asp, D) C4H5NO3 115.02694 115.0886 3.9–4.5 (4) 5.3

Cysteine (Cys, C) C3H5NOS 103.00919 103.1448 8.3–9.5 (9) 1.7

Glutamic acid (Glu, E) C5H7NO3 129.04259 129.1155 4.3–4.5 (4.5) 6.2

Glutamine (Gln, Q) C5H8N2O2 128.05858 128.1308 — 4.0

Glycine (Gly, G) C2H3NO 57.02146 57.0520 — 7.2

Histidine (His, H) C6H7N3O 137.05891 137.1412 6.0–7.0 (6.3) 2.2

Isoleucine (Ile, I) C6H11NO 113.08406 113.1595 — 5.2

Leucine (Leu, L) C6H11NO 113.08406 113.1595 — 9.0

Lysine (Lys, K) C6H12N2O 128.09496 128.1742 10.4–11.1 (10.4) 5.7

Methionine (Met, M) C5H9NOS 131.04049 131.1986 — 2.4

Phenylalanine (Phe, F) C9H9NO 147.06841 147.1766 — 3.9

Proline (Pro, P) C5H7NO 97.05276 97.1167 — 5.1

Serine (Ser, S) C3H5NO2 87.03203 87.0782 — 6.9

Threonine (Thr, T) C4H7NO2 101.04768 101.1051 — 5.8

Tryptophan (Trp, W) C11H10N2O 186.07931 186.2133 — 1.3

Tyrosine (Tyr, Y) C9H9NO2 163.06333 163.1760 9.7–10.1 (10.0) 3.2

Valine (Val, V) C5H9NO 99.06841 99.1326 — 6.6

aFor corresponding structures, see Figure A.1C.2 and Figure A.1C.4.bThe molecular mass of a normally terminated and unmodified peptide or protein may be calculated by summing the masses of theappropriate amino acid residues and adding the masses of H and OH for the N and C termini, respectively. In cases where cysteines arelinked to form disulfide bridges, the mass of two hydrogen atoms should be subtracted for each disulfide bridge in the molecule.Specifically, monoisotopic masses were calculated using the atomic masses of the most abundant isotope of the elements: C = 12.0000000,H = 1.0078250, N = 14.0030740, O = 15.9949146, and S = 31.9720718. Average masses were calculated using the atomic weights ofthe elements: C = 12.011, H = 1.00794, N = 14.00674, O = 15.9994, and S = 32.066.cThese values are included for anyone wishing to make approximate isoelectric point determinations based on protein composition.Values for the terminal residues depend on the identity of the residue: α-amino, pKa 6.8–8.2 (8.0); α-carboxyl, pKa 3.2–4.3 (3.6). Valuesin parentheses are based on those given by Matthew et al. (1978) and provide a good starting point for determinations.


A.1C.10


Table A.1C.4 Mass Changes Due to Some Post-Translational Modifications of Peptidesand Proteinsa

Modificationb Monoisotopic mass change

Average mass change

Common modificationsPyroglutamic acid formation from Gln −17.0265 −17.0306

Disulfide bond (cystine) formation −2.0157 −2.0159

C-terminal amide formation from Gly −0.9840 −0.9847

Deamidation of Asn and Gln −0.9840 −0.9847

Methylation 14.0157 14.0269

Hydroxylation 15.9949 15.9994

Oxidation of Met 15.9949 15.9994

Proteolysis of a single peptide bond 18.0106 18.0153

Formylation 27.9949 28.0104

Acetylation 42.0106 42.0373

Carboxylation of Asp and Glu 43.9898 44.0098

Phosphorylation 79.9663 79.9799

Sulfation 79.9568 80.0642

Cysteinylation 119.0041 119.1442

Glycosylation with pentoses (Ara, Rib, Xyl) 132.0423 132.1161

Glycosylation with deoxyhexoses (Fuc, Rha) 146.0579 146.1430

Glycosylation with hexosamines (GalN, GlcN) 161.0688 161.1577

Glycosylation with hexoses (Fru, Gal, Glc, Man) 162.0528 162.1424

Modification with lipoic acid (amide bond to lysine) 188.0330 188.3147

Glycosylation with N-acetylhexosamines (GalNAc, GlcNAc) 203.0794 203.1950

Farnesylation 204.1878 204.3556

Myristoylation 210.1984 210.3598

Biotinylation (amide bond to lysine) 226.0776 226.2994

Modification with pyridoxal phosphate (Schiff base to lysine) 231.0297 231.1449

Palmitoylation 238.2297 238.4136

Stearoylation 266.2610 266.4674

Geranylgeranlylation 272.2504 272.4741

Glycosylation with N-acetylneuraminic acid (sialic acid, NeuAc, NANA, SA)

291.0954 291.2579

Glutathionylation 305.0682 305.3117

Glycosylation with N-glycolylneuraminic acid (NeuGe) 307.0903 307.2573

5′-Adenosylation 329.0525 329.2091

Modification with 4′-phosphopantetheine 339.0780 339.3294

ADP-ribosylation (from NAD) 541.0611 541.3052

Adventitious modificationsAcrylamide 71.0371 71.0788

Glutathione 304.0712 304.3038

2-Mercaptoethanol 75.9983 76.1192

aTo obtain the molecular mass of a modified peptide or protein, the appropriate mass changes should bealgebraically added to the molecular mass calculated for the unmodified molecule.bA more extensive list of modifications is available from the Delta mass site at http://www.medstv.unimelb.edu.au/WWWDOCS/SVIMRDocs/MassSpec/deltamassV2.html.


A.1C.11


LITERATURE CITED

Chothia, C. 1976. The nature of the accessible and buried surfaces in proteins. J. Mol. Biol. 105:1-14.

Dayhoff, M.O., Schwartz, R.M., and Orcutt, B.C. 1978. A model of evolutionary change in proteins. In Atlasof Protein Sequence and Structure (M. Dayhoff, ed.) Vol. 5, pp. 345-352. National Biomedical ResearchFoundation, Washington, D.C.

Frömmel, C. 1984. The apolar surface area of amino acids and its empirical correlation with hydrophobicfree energy. J. Theor. Biol. 111:247-260.

Rose, G.D., Geselowitz, A.R., Lesser, G.J., Lee, R.H., and Zehfus, M.H. 1985. Hydrophobicity of amino acidresidues in globular proteins. Science 229:834-838.

Matthew, J.B., Friend, S.H., Botelho, L.H., Lehman, L.D., Hanania, G.I.H., and Gurd, F.R.N. 1979. Biochem.Biophys. Res. Commun. 81:416-421.

Sambrook, J., Fritsch, E.F., and Maniatis, T.M. (eds.). 1989. Molecular Cloning: A Laboratory Manual, 2nded. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York.

Sharp, P.M., Cowe, E., Higgins, D.G., Shields, D.C., Wolfe, K.H., and Wright, F. 1988. Codon usage patternsin E. coli, B. subtilis, S. cerevisiae, S. pombe, D. melanogaster, and H. sapiens: A review of the considerablewithin-species diversity. Nucl. Acids Res. 16: 8207-8211.

Sweet, R.M. and Eisenberg, D. 1983. Correlation of sequence hydrophobicities measures similarity inthree-dimensional protein structure. J. Mol. Biol. 171:479-488.

Wada, K.-N., Aota, S.-I., Tsuchiya, R., Ishibashi, F., Gojobori, T., and Ikemura, T. 1990. Codon usagetabulated from the GenBank genetic sequence data. Nucl. Acids Res. 18 (Suppl.):2367-2411.

Contributed by Andrew Ellington and J. Michael Cherry (codon usage)Massachusetts General HospitalBoston, Massachusetts


A.1C.12


APPENDIX 1DCharacteristics of Nucleic AcidsNucleic acids have traditionally been regarded solely as informational macromoleculeswith limited secondary structural features, but recent discoveries have vastly expandedthe repertoire of polynucleotide structure and function. While this manual has in generalconcentrated on how to manipulate DNA and RNA, this section details their structuralfeatures. In addition, some experimentally useful properties of the mononucleotidebuilding blocks are listed in Table A.1D.1. The chemical structures of the mononu-cleotides can be seen in Figure A.1D.1, and aspects of nucleotide stereochemistry that areimportant to an understanding of base pairing and secondary structure can be found inFigure A.1D.2. Although Watson-Crick pairings play a critical role in defining nucleicacid secondary structures, a wide variety of alternative base pairings can be important inhigher order conformations; some of these are detailed in Figure A.1D.3. Finally, evengiven only Watson-Crick-style pairings, secondary structures with significantly differentfeatures can be formed. Figure A.1D.4 overviews the differences between A-, B-, andZ-form helices.

Table A.1D.1 Physical Characteristics of the Nucleotidesa

Nucleotide Mol. wt.(g/mol)

λmax(nm)

λmin(nm)

εmax(mM−1 cm−1)

A280/A260TLC mobilityb

A B C

ATP 507.2 259 227 15.4 0.15 0 6 34ADP 427.2 259 227 15.4 0.16 0 26 54AMP 347.2 259 227 15.4 0.16 11 52 65Adenosinec 267.2 260 227 14.9 0.14 — — —dATPd 491.2 259 226 15.4 0.15 0 — 35dAMPd 331.2 259 226 15.2 0.15 11 52 —dA 251.2 260 225 15.2 0.15 — — —

CTP 483.2 271 249 9.0 0.97 0 11 41CDP 403.2 271 249 9.1 0.98 0 33 64CMP 323.2 271 249 9.1 0.98 15 64 75Cytidine 243.2 271 250 9.1 0.93 — — —dCTPd 467.2 272 — 9.1 0.98 0 — 43dCMP 307.2 271 249 9.3 0.99 18 65 —dC 227.2 271 250 9.0 0.97 — — —

GTP 523.2 253 223 13.7 0.66 0 5 25GDP 443.2 253 224 13.7 0.66 0 17 45GMP 363.2 252 224 13.7 0.66 6 40 51Guanosinec 283.2 253 223 13.6 0.67 — — —dGTPd 507.2 252 222 13.7 0.66 0 — 26dGMPd 347.2 253 222 13.7 0.67 6 41 —dG 267.2 254 223 13.0 0.68 — — —

UTP 484.2 262 230 10.0 0.38 0 14 49UDP 404.2 262 230 10.0 0.39 0 41 71UMP 324.2 262 230 10.0 0.39 20 75 80Uridine 244.2 262 230 10.1 0.35 — — —

TTPd 482.2 267 — 9.6 0.73 0 — 52TMPd 322.2 267 234 9.6 0.73 24 74 —Thymidined 242.2 267 235 9.7 0.70 — — —a Spectral data are assembled from Fasman (1975) at pH 7.0 except where footnoted otherwise.b TLC mobility is expressed as the percent distance a given spot migrates relative to the solvent front (Rf) in three differentTLC systems using 0.5-mm polyethylenimine cellulose plates: “A” is 0.25 M LiCl, “B” is 1.0 M LiCl, and “C” is 1.6 MLiCl.c Spectral measurements taken at pH 6.0.d Spectral data assembled from Dawson et al. (1987).

Supplement 66

Current Protocols in Molecular Biology (1996) A.1D.1-A.1D.11Copyright © 2004 by John Wiley & Sons, Inc.

A.1D.1


R

N

N

N

AN N

H

H

RH

N

N

N N

H

G

O

N H

HR O

OH

UH HN

N

H

R O

H H

H N

N

C

N

dR O

OH3C

TH HN

N

O

HO

HO

O

base

HO OH

3 ′ 2 ′

4 ′ 1 ′

(04 ′)5 ′OOO

O O O

PP P

O–O–O–

pKa = 6 –O

pKa<1

pKa = 12.5

pKa = 9.4

pKa = 9.4pKa = 3.8

pKa = 2.4pKa<1

pKa = 4.4

guanosineadenosine uridine

cytidine ribonucleoside 5 ′ triphosphate

pKa 14thymidine

deoxyribonucleoside

5 6

1

2

H

349

8

H7

21

3

45

6

base

H

pKa = 10.0

αγ β

Figure A.1D.1 Line drawings of the nucleotides. Thechemical structure that predominates at neutral pH isshown. Drawings of the nucleotide bases and their associ-ated sugars, either ribose (R) or deoxyribose (dR), areshown separately. In the representations of ribose (as anucleoside triphosphate) and deoxyribose (as a nucleotide),the bold lines indicate that this portion of the sugar is comingout of the page toward the reader. In this view, the base isfound above the plane of the sugar, while the 3′ hydroxylgroup is found below the plane of the sugar.

The pKa values for all groups are shown; pKas above 7 implyproton dissociation from the pictured structure, while pKasbelow 7 imply proton association to the pictured structure.The tautomeric form of a given base may change at different

pH values. The pKa values given are for nucleotide mono-phosphates and were taken from Dawson et al. (1987); afuller discussion of the chemical basis for these values canbe found in a review by T’so (1974).

The small numbers adjacent to adenosine, uridine, andribose indicate the nomenclature of the purines, pyrimidi-nes, and sugars, respectively. Groups appended to a ringhave the same numbering as the position to which they arelinked; thus, the “O6” moiety of guanosine is the carbonyloxygen bonded to C6 in the ring. Similarly, “O3′” on riboseor deoxyribose indicates the oxygen of the hydroxyl groupbonded to C3′ in the ring. The α, β, and γ phosphates in anucleoside triphosphate are also indicated.


A.1D.2

O

H

O

O

N

H

N

N

H

H

HO

HO OH

N

N

O

NHO

HO OH

NN

N

H

H

O O

O

guanosine-anti guanosine-syn

base baseC5 ′ C5 ′

C3 ′ endo C2 ′ exo

C5 ′base

C3 ′ C2 ′

C2 ′C3 ′

C3 ′ endo–C2 ′ exo

N

H

C1 ′

N9

H

Figure A.1D.2 Nucleotide stereochemistry. Depending onthe rotation about the bond between C1′ of the sugar andeither N1 (for pyrimidines) or N9 (for purines), a nucleotidecan be described as either “anti” or “syn.” Because of stericconstraints, nucleotides are generally found in the “anti”configuration, with their Watson-Crick hydrogen bond do-nor-acceptors swung outward away from the plane of thesugar ring. However, guanosine is sometimes found in a“syn” configuration, both in polynucleotides and in solution.In this form, the bulk of the purine ring is positioned directlyover the plane of the sugar. The sugar ring can also adopt

different stereochemistries. These are labeled according towhich group is bent out of the plane of the ring, and in whichdirection. If a portion of the ring is bent “upward” toward thebase, this is known as “endo,” while if it is bent “downward”away from the base, this is known as “exo.” In the figure,plain lines represent bonds that are within the plane of thesugar, while bold lines indicate that the bond is bent out ofthe plane. Hence, “C3′ endo–C2′ exo” describes a furanosering in which the 2′ and 3′ carbons have been twisted inopposite directions and the bond connecting them crossesthe plane of the ring.


A.1D.3


H

RO

R

O HN H

U HH

N

N

AN N N

N

H

H

A:U

RO

R

HH

HH

N

N

N N N

N

H

G C

O N

N H

H

R

N

N

N

AN N

H

H

H

RO

HH

HH N

N

C

N

R

H

H

N

N

N N

H

G

O

N H

H

R

O H

U HN

N

O

G:C

Watson-Crick pairings

Wobble pairings

H

H

A:C G:U

H

O O

H

R

N

N

N

AN N

H

H

H

H

N

NHR

H

O O

H

R

N

N

N

AN N

H

H

H

H

N

NH R

U

Reverse Hoogsteen(antiparallel chains)

U

Hoogsteen(parallel chains)


A.1D.4

Characteristics ofNucleic Acids

O

R

O

RH

N

N

N N

H

G

N H

H

N

N

NG

N N

H

HH

H

G-anti:G-syn (antiparallel chains)

O

O

RN

N

H

HH

N

H

N

N

HR

N

N

H

H

H

N

HOR

N

H

H

H

N

N

N

N

N

N

H

N

H

H

H H

N

N

N

NdR

N

H N H

H

N

H

N

dR

N

NH

HN

H

N

H

NdR

O

O

HH N

H O

O

dR

N

N

N

C:G—G (as in tRNA Phe)

"G quartet"

Figure A.1D.3 Base pairing schemas. The chemical struc-tures of the nucleotide bases determine the formation ofsecondary and tertiary structures in nucleic acids. A widevariety of hydrogen bonding schemas (indicated by dashedlines) are possible between different bases. Watson-Crickpairings are perhaps the most widely known and are thebasis of the double helical structure of complementary,anti-parallel DNA strands. Other base pairs can also beaccommodated within the double helix, such as “wobblepairings,” in which the bases are slightly off-center withrespect to each other. By using the N7 hydrogen bond

acceptor of the purine bases adenosine and guanosine, aneven wider variety of structures becomes possible, includingHoogsteen base pairs and a G-G pairing in which one of theguanosine residues assumes a “syn” conformation. Bondsinvolving N7 of the purine bases allow tertiary structuralinteractions to occur in nucleic acids, including triple basepairs (such as those found in tRNA) and the recently de-scribed “G quartet” (Sen and Gilbert, 1988). A discussion ofthe structural possibilities of base pairing can be found inSaenger’s superlative book, Principles of Nucleic AcidStructure (1993).


A.1D.5

Figure A.1D.4 Nucleic acid secondary structures. Thestructural consequence of the ability of nucleotides to formWatson-Crick base pairs is nucleic acid double helices. In thisfigure, the self-complementary 12-mer CGCGAATTCGCG isshown as both A- and B-form helices. Two representationsof the A helix have been shown in order to emphasize thedepth of the major groove. The arrows and brackets in thesefigures are not drawn to scale.

While both of these helices are right-handed (in terms ofanthropomorphic referents, if you were to point your thumbalong a strand in a 5′ to 3′ manner, the twist of the helixwould be the same as the curl of your right hand), theirstructural details are very different: B DNA has roughly 10bases per full turn, while A DNA and A RNA have 11 to 12;the major groove of B-form helices is wide and the minorgroove is narrow, while for A-form helices this is reversed; in


A.1D.6

B-forms the base pairs are located close to the helix axis(as can be seen in end-on views), while in A-forms the basepairs are pushed out away from the long helical axis, leavinga “hole” in the middle of the polynucleotide coil (if oneimagines DNA as a flat ribbon, then B DNA is twisted fromits ends, while A DNA is coiled on itself).

Different helical forms are largely due to differences in sugarstereochemistry. Examples of a 2′ endo deoxyribose (foundin B DNA) and a 3′ endo deoxyribose (found in A DNA) areindicated.

While there are a variety of other helical forms, the most

striking is that found in Z DNA. The Z DNA coil is left- ratherthan right-handed and contains G:C base pairs where theG is in the “syn” conformation (shown in the inset).

The uneven progression, or zigzag, of Z DNA can be moreeasily seen when the polynucleotide backbone is shown inisolation; the inset shows the connectivity between phos-phates by 5′ to 3′ vector arrows. Because of its odd shape,base pairs actually protrude from what would be a cavity inA or B DNA; thus, Z DNA has a minor but no major groove.This diagram is based on the original structure of alternatingC:G/G:C base pairs (Wang et al., 1979).


A.1D.7


A.1D.8



A.1D.9



A.1D.10


LITERATURE CITED

Dawson, M.C., Elliott, D.C., Elliott, W.H., and Jones, K.M. (eds.). 1987. Data for Biochemical Research,3rd ed. Clarendon Press, Oxford.

Fasman, G. (ed.). 1975. Handbook of Biochemistry and Molecular Biology, Vol. 1: Nucleic Acids, 3rd ed.CRC Press, Boca Raton, Fla.

Saenger, W. 1993. Principles of Nucleic Acid Structure. Springer-Verlag, New York.

Sen, D. and Gilbert, W. 1988. Formation of parallel four-stranded complexes by guanine-rich motifs inDNA and its implications for meiosis. Nature 334:364-366.

T’so, P.O.P. 1974. Bases, nucleosides, and nucleotides. In Basic Principles in Nucleic Acid Chemistry, Vol.1 (P.O.P. T’so, ed.) pp. 453-584. Academic Press, San Diego.

Wang, A.H., Quigley, G.J., Kolpak, F.J., Crawford, J.L., van Boom, J.H., van der Marel, G., and Rich, A.1979. Molecular structure of a left-handed double helical DNA fragment at atomic resolution. Nature282:680-686.


A.1D.11


APPENDIX 1ERadioactivity

ABSTRACT

This appendix provides selected properties of radioisotopes commonly used in themolecular biology laboratory. Curr. Protoc. Mol. Biol. 79:A.1E.1-A.1E.5. C© 2007 byJohn Wiley & Sons, Inc.

Keywords: radioactivity isotope counting efficiency

INTRODUCTION

When working with radioactivity, it is important to have a clear understanding of theproperties of the radioisotopes and their decay. Table A.1E.1 lists physical characteristicsof radioisotopes that are commonly used in the molecular biology laboratory. A discussionof the different types of emissions from radioisotopes can be found in APPENDIX 1F, whichprovides a thorough discussion of safety considerations for working with radioactivity.For general comparison, Table A.1E.2 illustrates relative shielding capabilities of variousmaterials. The discussion in this unit focuses on aspects of radioactive decay that areimportant in quantitative analyses.

Table A.1E.1 Physical Characteristics of Commonly Used Radionuclidesa

Nuclide Emission Half-lifeDecay constant

(kdecay)Energy, max(MeV)

Range ofemission, max

Approx. specificactivity at 100%enrichment (Ci/mg)

Decayproduct

3H β 12.43 years 0.056 year−1 0.0186 0.42 cm (air) 9.6 32 He

14C β 5370 years 0.156 21.8 cm (air) 4.4 mCi/mg 147 N

32Pb β 14.3 days 0.0485 day−1 1.71 610 cm (air) 285 3216 S

0.8 cm (water)

0.76 cm(Plexiglas)

33Pb β 25.4 days 0.249 49 cm 156 3316 S

35S β 87.4 days 0.079 day−1 0.167 24.4 cm (air) 43 3517 Cl

125Ic γ 60 days 0.0116 day−1 0.027–0.035 0.2 mm (lead) 14.2 12552 Te

131Ic β 8.04 days 0.606 165 cm (air) 123 13054 Xe

γ 0.364 2.4 cm (lead)aTable compiled based on information in Lederer et al. (1967) and Shleien (1987).bRecommended shielding is Plexiglas; half-value layer measurement is 1 cm.cRecommended shielding is lead; half-value layer measurement is 0.02 mm.

Current Protocols in Molecular Biology A.1E.1-A.1E.5, July 2007Published online July 2007 in Wiley Interscience (www.interscience.wiley.com).DOI: 10.1002/0471142727.mba01es79Copyright C© 2007 John Wiley & Sons, Inc.


A.1E.1

Supplement 79

Radioactivity

A.1E.2


Table A.1E.2 Shielding Radioactive Emissiona

β emitters

Thickness (mm) to reduce intensity by 50%

Energy(MeV)

Mass (mg)/cm2 to reduceintensity by 50%

Water Glass Lead Plexiglas

0.1 1.3 0.013 0.005 0.0011 0.0125

1.0 48 0.48 0.192 0.042 0.38

2.0 130 1.3 0.52 0.115 1.1

5.0 400 4.0 1.6 0.35 4.2

γ emitters

Thickness of material (cm) to attenuate a broad beam ofγ-rays by a factor of 10

Energy(MeV)

Water Aluminum Iron Lead

0.5 54.6 20.3 6.1 1.8

1.0 70.0 24.4 8.2 3.8

2.0 76.0 32.0 11.0 5.9

3.0 89.0 37.0 12.0 6.4aFrom Dawson et al. (1986). Reprinted with permission.

NOTE: It is important for the researcher to be familiar with applicable regulations andapproved procedures for the safe use of radioisotopes. Because the real hazards of lowlevels of radiation are not known, it is generally assumed that any unavoidable exposureis too much.

RADIOACTIVE DECAY

Radioactive decay is an entirely random process. The probability of decay during anyparticular interval is the same as the probability of decay during any other interval.Starting with N0 radioactive atoms, the number remaining at time t is:

N0 = Nt × e−kdecayt

where kdecay is the rate constant of decay in units of inverse time. The half-life (t1/2) isthe time it takes for half of the isotope to decay. Each radioisotope has a characteristicvalue of kdecay and thus a characteristic half-life (Table A.1E.1).

The correlation between half-life and remaining radioactivity at any given time is il-lustrated in Figure A.1E.1. Radioactive decay can be calculated from a date when theconcentration and specific radioactivity were known using the above equation (i.e., wherethe fraction remaining is Nt/N0), or can be extrapolated from decay tables for specificisotopes (Table A.1E.3).

Calculations of radioactive decay are straightforward only when each molecule is labeledwith a single radioactive isotope, as is usually the case. If a molecule is labeled withseveral radioactive isotopes, the effective half-life is shorter. If only a fraction of themolecules are labeled with a radioactive isotope, then the decay formula only applies tothe labeled portion of the mixture, as the concentration of the unlabeled compound neverchanges.


A.1E.3


Table A.1E.3 Decay factors for calculating the amount of radioactivity present at a given time after a refer-ence date. for example, a vial containing 1.85 MBq (50 µCi) of an 35S-labeled compound on the referencedate will have the following activity 33 days later: 1.85 × 0.770 = 1.42 MBq; 50 × 0.770 = 38.5 µCi

125I Half-Life: 60.0 days 32P Half-Life: 14.3 daysDays Hours

0 2 4 6 8 10 12 14 16 18 0 12 24 36 48 60 72 840 1.000 0.977 0.955 0.933 0.912 0.891 0.871 0.851 0.831 0.812 0 1.000 0.976 0.953 0.930 0.908 0.886 0.865 0.844

Day

s20 0.794 0.776 0.758 0.741 0.724 0.707 0.691 0.675 0.660 0.645 D

ays

4 0.824 0.804 0.785 0.766 0.748 0.730 0.712 0.69540 0.630 0.616 0.602 0.588 0.574 0.561 0.548 0.536 0.524 0.512 8 0.679 0.662 0.646 0.631 0.616 0.601 0.587 0.57360 0.500 0.489 0.477 0.467 0.456 0.445 0.435 0.425 0.416 0.406 12 0.559 0.546 0.533 0.520 0.507 0.495 0.483 0.47280 0.397 0.388 0.379 0.370 0.362 0.354 0.345 0.338 0.330 0.322 16 0.460 0.449 0.439 0.428 0.418 0.408 0.398 0.389

100 0.315 0.308 0.301 0.294 0.287 0.281 0.274 0.268 0.262 0.256 20 0.379 0.370 0.361 0.353 0.344 0.336 0.328 0.320120 0.250 0.244 0.239 0.233 0.228 0.223 0.218 0.213 0.208 0.203 24 0.312 0.305 0.298 0.291 0.284 0.277 0.270 0.264140 0.198 0.194 0.189 0.185 0.181 0.177 0.173 0.169 0.165 0.161 28 0.257 0.251 0.245 0.239 0.234 0.228 0.223 0.217160 0.157 0.154 0.150 0.147 0.144 0.140 0.137 0.134 0.131 0.128 32 0.212 0.207 0.202 0.197 0.192 0.188 0.183 0.179180 0.125 0.122 0.119 0.117 0.114 0.111 0.109 0.106 0.104 0.102 36 0.175 0.170 0.166 0.162 0.159 0.155 0.151 0.147200 0.099 0.097 0.095 0.093 0.090 0.088 0.086 0.084 0.082 0.081 40 0.144 0.140 0.137 0.134 0.131 0.127 0.124 0.121220 0.079 0.077 0.075 0.073 0.072 0.070 0.069 0.067 0.065 0.064 44 0.119 0.116 0.113 0.110 0.108 0.105 0.102 0.100240 0.063 0.061 0.060 0.058 0.057 0.056 0.054 0.053 0.052 0.051 48 0.098 0.095 0.093 0.091 0.089 0.086 0.084 0.082

52 0.080 0.078 0.077 0.075 0.073 0.071 0.070 0.068

131I Half-Life: 8.04 days 35S Half-Life: 87.4 daysHours Days

0 6 12 18 24 30 36 42 48 54 60 66 0 1 2 3 4 5 60 1.000 0.979 0.958 0.937 0.917 0.898 0.879 0.860 0.842 0.824 0.806 0.789 0 1.000 0.992 0.984 0.976 0.969 0.961 0.954

Day

s

3 0.772 0.756 0.740 0.724 0.708 0.693 0.678 0.664 0.650 0.636 0.622 0.609 1 0.946 0.939 0.931 0.924 0.916 0.909 0.9026 0.596 0.583 0.571 0.559 0.547 0.533 0.524 0.513 0.502 0.491 0.481 0.470 W

eeks

2 0.895 0.888 0.881 0.874 0.867 0.860 0.8539 0.460 0.450 0.441 0.431 0.422 0.413 0.405 0.396 0.387 0.379 0.371 0.363 3 0.847 0.840 0.833 0.827 0.820 0.814 0.807

12 0.355 0.348 0.340 0.333 0.326 0.319 0.312 0.306 0.299 0.293 0.286 0.280 4 0.801 0.795 0.788 0.782 0.776 0.770 0.76415 0.274 0.269 0.263 0.257 0.252 0.246 0.241 0.236 0.231 0.226 0.221 0.216 5 0.758 0.752 0.746 0.740 0.734 0.728 0.72218 0.212 0.207 0.203 0.199 0.194 0.190 0.186 0.182 0.178 0.175 0.171 0.167 6 0.717 0.711 0.705 0.700 0.694 0.689 0.68321 0.164 0.160 0.157 0.153 0.150 0.147 0.144 0.141 0.138 0.135 0.132 0.129 7 0.678 0.673 0.667 0.662 0.657 0.652 0.64624 0.126 0.124 0.121 0.118 0.116 0.113 0.111 0.109 0.106 0.104 0.102 0.100 8 0.641 0.636 0.631 0.626 0.621 0.616 0.61227 0.098 0.095 0.093 0.091 0.089 0.088 0.086 0.084 0.082 0.080 0.079 0.077 9 0.607 0.602 0.597 0.592 0.588 0.583 0.57930 0.075 0.074 0.072 0.071 0.069 0.068 0.066 0.065 0.064 0.063 0.061 0.059 10 0.574 0.569 0.565 0.560 0.556 0.552 0.54733 0.058 0.057 0.056 0.054 0.053 0.052 0.051 0.050 0.049 0.048 0.047 0.046 11 0.543 0.539 0.534 0.530 0.526 0.522 0.51836 0.045 0.044 0.043 0.042 0.041 0.040 0.039 0.039 0.038 0.037 0.036 0.035 12 0.514 0.510 0.506 0.502 0.498 0.494 0.490

33P Half-Life: 25.4 daysDays

0 1 2 3 4 5 6 7 8 90 1.000 0.973 0.947 0.921 0.897 0.872 0.849 0.826 0.804 0.782

Day

s

10 0.761 0.741 0.721 0.701 0.683 0.664 0.646 0.629 0.612 0.59520 0.579 0.564 0.549 0.534 0.520 0.506 0.492 0.479 0.466 0.45330 0.441 0.429 0.418 0.406 0.395 0.385 0.374 0.364 0.355 0.34540 0.336 0.327 0.318 0.309 0.301 0.293 0.285 0.277 0.270 0.26350 0.256 0.249 0.242 0.236 0.229 0.223 0.217 0.211 0.205 0.20060 0.195 0.189 0.184 0.179 0.174 0.170 0.165 0.161 0.156 0.15270 0.148 0.144 0.140 0.136 0.133 0.129 0.126 0.122 0.119 0.11680 0.113 0.110 0.107 0.104 0.101 0.098 0.096 0.093 0.091 0.08890 0.086 0.084 0.081 0.079 0.077 0.075 0.073 0.071 0.069 0.067

100 0.065 0.064 0.062 0.060 0.059 0.057 0.055 0.054 0.053 0.051110 0.050 0.048 0.047 0.046 0.045 0.043 0.042 0.041 0.040 0.039120 0.038 0.037 0.036 0.035 0.034 0.033 0.032 0.031 0.030 0.030

Figure A.1E.1 Correlation of loss of radioactivity with elapsing half-lives of an isotope.

Radioactivity

A.1E.4


COUNTING EFFICIENCY

The output for any radioactivity counter is given in counts (or counts per minute, cpm),which are less than the actual disintegrations from the radioisotope. Thus, it is impor-tant to take into account the counting efficiency, which is the fraction of radioactivedisintegrations detected by the counter. Efficiency is determined by counting a standardsample under conditions identical to those used in the experiment. The efficiency for125I is typically >90%, and is dependent on the geometry of the instrument. Becausethe detector doesn’t entirely surround the tube, a few γ rays (photons) will miss thedetector. The efficiency for 32P and 35S is typically ∼80%. With 3H, the efficiencyis much lower, usually 40% to 50%. This is mostly a consequence of the physics of3H decay, which can release energy as electrons (only some of which have sufficientenergy to be detected) or as neutrinos (which are not detected). The efficiency can-not be improved by better instrumentation or better scintillation fluid. In addition, thecounting efficiency for 3H is reduced by the presence of any color in the countingtubes, by nonhomogeneous mixing of water and scintillation fluid, or by radioactivitythat is trapped in tissue (because emitted electrons do not travel into the scintillationfluid).

SPECIFIC RADIOACTIVITY

The packaging label on radioactive compounds usually states the specific radioactivityas curies per millimole (Ci/mmol). Because measurements are expressed in cpm, it isoften more convenient to express the specific radioactivity as cpm/fmol. Ci/mmol canbe converted to cpm/fmol using the conversion factors in Table A.1E.4. For example,if counting efficiency is 85%, a specific activity of 2500 Ci/mmol is equivalent to 4718cpm/fmol. In many countries, specific radioactivity is provided in GBq/mmol, which canalso be converted to cpm/fmol.

COUNTING ERROR

Because decay is random, it is subject to sampling error. The cpm measured in a samplerepresents an average, with more counts in some minutes and fewer in others, and adistribution of counts that follows a Poisson distribution. There is no way to know the“real” number of counts, but a range of counts can be calculated that is 95% certain tocontain the true average value. This range is known as the 95% confidence interval. ThePoisson distribution explains why it is helpful to count samples longer when the numberof counts is small. As an example, Table A.1E.5 shows that the confidence interval isnarrower when longer counting times are used.

LITERATURE CITED

Dawson, R.M.C., Elliot, D.D., Elliot, W.H., and Jones, K.M. (eds.) 1986. Data for Biochemical Research.Alden Press, London.

Lederer, C.M., Hollander, J.M., and Perlman, I. (eds.) 1967. Table of Radioisotopes, 6th ed. John Wiley &Sons, New York.

Shleien, B. (ed.) 1987. Radiation Safety Manual for Users of Radioisotopes in Research and AcademicInstitutions. Nucleon Lectern Associates, Olney, Maryland.


A.1E.5


Table A.1E.4 Conversion Factors for Radioactivity

Measurement of Radioactivity

The SI unit for measurement of radioactivity is the becquerel:

1 Bq = 1 disintegration per second

The more commonly encountered unit is the curie (Ci):

1 Ci = 3.7 × 1010 Bq

= 2.22 × 1012 disintegrations per minute (dpm)

1 millicurie (mCi) = 3.7 × 107 Bq = 2.22 × 109 dpm

1 microcurie (µCi) = 3.7 × 104 Bq = 2.22 × 106 dpm

Conversion factors:

1 day = 1.44 × 103 min = 8.64 × 104 sec

1 year = 5.26 × 105 min = 3.16 × 107sec

counts per minute (cpm) = dpm × (counting efficiency)

Measurement of Dose

The SI unit for energy absorbed from radiation is the gray (Gy):

1 Gy = 1 joule/kg

Older units of absorbed energy are the rad (r) and roentgen (R):

1 r = 100 ergs/g = 10−2 Gy

1 R = 0.877 r in air = 0.93 − 0.98 r in water and tissue

The SI unit for radiation dosage is the sievert (Sv), which takes

into account the empirically determined relative biological

effectiveness (RBE) of a given form of radiation:

dosage [Sv] = RBE × dosage [Gy]

RBE = (biological effect of a dose of standard radiation[Gy])(biological effect of a dose of other radiation[Gy])

RBE = 1 for commonly encountered radionuclides

The older unit for dosage is the rem (roentgen-equivalent-man):

1 rem = 0.01 Sv

Table A.1E.5 Example of Confidence Intervals Using Different Counting Times

1 min 10 min 100 min

Cpm 100 100 100

Total counts 100 1000 10000

95% CI of counts 81.4–121.6 938–1062 9804–10196

95% CI of cpm 81.4–121.6 93.8–106.2 98.0–102.0

APPENDIX 1FSafe Use of Radioisotopes

Jill Meisenhelder1 and Steve Bursik1

1The Salk Institute, La Jolla, California

ABSTRACT

The pursuit of scientific knowledge has been considerably advanced by the use of biochemicalmolecules that incorporate radioisotopes at specific sites. The fate of these labeled molecules,and/or the radiolabeled products that result from biochemical reactions in which the parentmolecule was involved, can be traced using a variety of instruments that detect radioactivity.This appendix begins with a discussion of the principles of radioactivity in order to providethe reader/user with knowledge on which to base a common sense approach to the safe use ofisotopes. The characteristics of isotopes most commonly used in a molecular biology laboratoryare then detailed, as well as the safety precautions and monitoring methods peculiar to each one.Detection and imaging methods used in experimental analysis are reviewed. Finally, an outlineof an orderly response to a spill of radioactive material is presented. Curr. Protoc. Mol. Biol.79:A.1F.1-A.1F.18. C© 2007 by John Wiley & Sons, Inc.

Keywords: radiation safety radioactivity isotopes decay shielding monitoring

exposure dosimeter

INTRODUCTIONThe use of radioisotopes to label specific

molecules in a defined way has greatly fur-thered the discovery and dissection of bio-chemical pathways. The development of meth-ods to inexpensively synthesize such taggedbiological compounds on an industrial scalehas enabled them to be used routinely in labo-ratory protocols, including many detailed inCurrent Protocols. Although most of theseprocedures involve the use of only microcurie(µCi) amounts of radioactivity, some (partic-ularly those describing the metabolic labelingof proteins or nucleic acids within cells) canrequire amounts on the order of tens of milli-curies (mCi). In all cases where radioisotopesare used, depending on the quantity and na-ture of the isotope, certain precautions mustbe taken to ensure the safety of everyone inthe laboratory. This unit outlines a few suchconsiderations relevant to the isotopes mostfrequently used in biological research.

In designing safe protocols for the use of ra-dioactivity, the importance of common sense,based on an understanding of the general prin-ciples of radioactive decay, and the importanceof continuous monitoring with a hand-held ra-diation monitor (e.g., Geiger counter), cannotbe overemphasized. In addition, it is critical totake into account the relevant and applicablerules, regulations, and limits of exposure. Al-though different countries have different rules,

compliance is not optional: an institution’slicense to use radioactivity normally dependson strict adherence to such rules.

BACKGROUND INFORMATION

The Radioactive Decay ProcessAs anyone who has taken a basic chemistry

course will remember, each element is char-acterized by its atomic number (Z), defined asthe number of protons in the nucleus of thatatom. Z is therefore unique to each elementand determines the identification and chem-istry of that particular element. Isotopes of agiven element exist because some atoms ofeach element, while by definition having thesame number of protons, have a different num-ber of neutrons and therefore a different atomicweight or atomic mass number (A), which in-dicates the total number of nucleons (protons+ neutrons). It should be noted that generally,the number of electrons outside the nucleusremains the same for all isotopes of a givenelement, so all isotopes of a given elementare equivalent with respect to their chemicalreactivity.

Radioactive decay is defined as the spon-taneous change in the structure of an atomaccompanied by the emission of energy. Thisoften results in the change of an atom of oneelement into the atom of a totally different ele-ment, a process termed nuclear transmutation,

Current Protocols in Molecular Biology A.1F.1-A.1F.18, July 2007Published online July 2007 in Wiley Interscience (www.interscience.wiley.com).DOI: 10.1002/0471142727.mba01fs79Copyright C© 2007 John Wiley & Sons, Inc.


A.1F.1

Supplement 79

Safe Use ofRadioisotopes

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as the number of protons (or neutrons) in theatom changes after decay. The energy releasedcan be particulate (i.e., α and β particles) ornon-particulate (i.e., γ- and X-rays). These arethe primary types of radiation encountered inbiological research. Emitted radiation is usu-ally measured in units of keV (kilo-electronvolts) or MeV (mega-electron volts).

An α particle is essentially the nucleus ofa helium atom, or two protons plus two neu-trons. They are relatively large, heavy parti-cles that move relatively slowly (compared toa β particle having the same amount of en-ergy). Containing two protons, the α parti-cle has a positive charge of 2+. With a rel-atively high electronic charge, it only travelsshort distances before it readily interacts withsome other atom via coulombic forces. Emit-ted α particles have discrete energies and areemitted from isotopes having high mass nuclei(atomic number Z > 82; e.g., thorium or ura-nium); such isotopes are not commonly usedin biological research except for specific ap-plications in electron microscopy and X-raydiffraction studies.

In contrast to α particles, β particles arelight, high-speed, singly charged particles.Negatively charged β particles are essentiallyelectrons of nuclear origin emitted when an in-tranuclear neutron changes to a proton with theattendant release of a neutrino (which is veryweakly interacting). Release of a β particlethus changes the atomic number and elemen-tal status of the isotope. β radiation is emittedacross a spectrum of different energies, themost energetic being stated as Emax. The aver-age energy can be approximated as Eave = 1/3Emax. An example is 32P, with an Emax of 1.71MeV and an Eave of 0.57 MeV. The neutrinois emitted with an energy equal to the differ-ence between the actual emitted β energy andEmax. Most of the β emitters used in the biol-ogy laboratory are “pure β emitters,” meaningthere is no other radiation emitted besides theβ particle and the neutrino.

γ radiation exhibits both particle and waveproperties, and its wavelength falls within therange of X-ray wavelengths. Physically, thereis no difference between γ and X-ray radia-tion. The only difference is their physical ori-gin. γ radiation is defined as that originatingfrom an atomic nucleus, while X-ray radiationoriginates from the electron cloud surround-ing the nucleus. Unlike β particle emission,the emission of γ radiation by itself producesan isotopic change rather than an elementalone; however, the resultant nuclei may be un-

stable and decay further, possibly releasingβ particles. γ radiation is emitted with adiscrete energy.

Isotopic decay may involve a chain or se-quence of events rather than just a single decay.Subsequent daughter products may also be ra-dioactive (unstable) and thus pose a hazard toworkers.

Following their emission, α and β radiationtravel varying distances at varying speeds, de-pending on their initial energy and the atomicnumber of the material through which they aremoving. The distance they actually travel be-fore interacting with the electrons or nuclei ofanother atom is termed their range and is adefined value for each kind of material. Thisrange is usually expressed as a maximum foreach type of particle, the particle’s energy, andmaterial. The energy and type of particulateradiation released (and therefore its potentialrange) dictates what type of shielding, if any,is necessary for protection against the radia-tion generated by the decay of the isotope. Asdiscussed previously, α radiation, even with ahigh amount of energy, will not penetrate veryfar into common materials. In fact, a sheetof paper can block most α radiation. There-fore, α radiation is not considered an externalhazard, the major concern being internal ex-posure. Theoretically, γ and X-ray radiationcan travel forever and do not have a specificrange. Practically, however, there is a measur-able decrease in the intensity of γ radiation asit penetrates greater thicknesses of material.This decrease is usually expressed as the half-value layer (HVL) or tenth-value layer (TVL)of a specific material (e.g., lead) and is used tocalculate the needed thickness of shielding.

When high-energy β particles released dur-ing the decay of 32P encounter the nuclei ofatoms with a high atomic number, a coulombicinteraction occurs. The β particle deceleratesand loses energy in the form of X-rays. SuchX-rays are termed bremsstrahlung radiation(German for “breaking radiation”); they aredetectable using most radiation survey meters,especially those designed for the detection ofγ or X-rays. The amount of bremsstrahlungradiation produced is directly proportional toboth the energy of the incident β particle andthe atomic number, Z, of the absorber. Hence,much more bremsstrahlung is produced by 32Pβ radiation than 35S β radiation (Emax for 35S is0.167 MeV) when incident on the same mate-rial, and more is produced by 32P β radiationwhen incident on a high-Z material such aslead compared to plastic.


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α, β, and γ emissions all have the poten-tial, upon encountering an atom, to interactwith and ionize the atom. Thus, these threetypes of emissions are called ionizing radia-tion. The formation of such ions may resultin the perturbation of biochemical processes:therein lies the health concern associated withradioactivity!

Units Used for RadioactivityThere are several measurable properties

used to describe the amount and physical ef-fects of ionizing radiation: activity, exposure,dose and dose equivalent.

Activity is the amount of radioactive mate-rial in a sample and it is measured in units ofcuries (Ci) or becquerels (Bq). A curie by def-inition is that amount of radioactive materialthat will produce 3.7 × 1010 disintegrations(ions) per second. This was originally deter-mined as the number of disintegrations thatoccur during the radioactive decay of 1 g ofradium-226. The becquerel is defined as 1 dis-integration per second. In biological research,the curie is a very large unit and the becquerela very small unit, so prefixes are added tothese roots to express the activity amounts be-tween these two extremes that are commonlyused, i.e., microcurie (µCi), Megabecquerel(MBq). Several useful tables on radioactivityand physical characteristics of radioisotopescan be found in APPENDIX 1E.

Exposure is defined as that amount of ion-ization (measured as electrical charge) pro-duced in a particular volume of air at standardtemperature and pressure (STP) by the passageof γ or X-ray radiation. This unit is only de-fined for γ or X-ray radiation. The exposureunit is the X unit, which is slowly supplant-ing the roentgen (R) for expressing this prop-erty. One X unit is 1 coulomb per kilogramof air. This property can be measured directlywith ionization chamber-based radiation sur-vey meters.

Dose is the property that describes the de-position of energy. It is defined as the amountof deposited radiation energy per mass unit ofabsorber. A deposition of 100 ergs in 1 gram ofmaterial is equivalent to 1 rad. There are 100rads in 1 gray, another common unit of dose.Defined doses for isotopes commonly used inthe molecular biology laboratory are shownin Table A.1F.1. Dose is important, but onlyconsiders physical factors.

The property of dose equivalent is most cor-rectly used to describe the potential for damageto an irradiated individual. The unit for doseequivalent is the rem. The number of rems is

obtained by multiplying the number of rads bya “quality factor,” which is based on the typeof ionizing radiation delivering the dose. Forβ particles and γ or X-rays this factor is 1 andtherefore rems and rads are equal for β radia-tion. In contrast, the quality factor associatedwith α particles is 20, so a dose of 1 rad dueto α particles would be recorded as 20 rem.Thus, dose equivalent is based on both physi-cal and biological factors. The dose equivalentis the most meaningful quantity used for radi-ation protection purposes and is the unit usedto record dosimeter badge readings. There are100 rems in a sievert, the unit of dose equiva-lent used in most countries other than the U.S.

Measuring Exposure to IonizingRadiation

The radiation dose received by materials(cells, scientists, etc.) near a radioactive sourcedepends not only on the specific type and en-ergy of the radiation absorbed, but also on thesubject’s distance from the source, the exis-tence of any intervening layers of attenuat-ing material (shielding, clothing, etc.), and thelength of time spent in the vicinity of the radi-ation source. To best measure doses to person-nel, everyone working with or in close prox-imity to radioactive sources should wear theappropriate type of radiation dosimeter badge(in addition to using a portable radiation mon-itor that can give an immediate indication ofthe presence of radiation). This is normallya requirement (not an option) for compliancewith an institution’s radioactive materials li-cense. Such badges are usually furnished bythe radiation safety department, collected atregular intervals, and sent for processing toa contracted company. Most institutions useeither TLDs (thermoluminescent dosimeters)or OSLDs (optically stimulated luminescentdosimeters). Both devices use crystals of cal-cium fluoride, lithium fluoride, or aluminumoxide; the electron structure of these crys-talline materials is altered following exposureto ionizing radiation. During processing, stim-ulation by either heat (for TLDs) or laser light(for OSLDs) will cause the crystals to lumi-nesce, and the intensity of luminescence isdirectly proportional to the dose of ionizingradiation that has been absorbed. The resultsare then compared with known controls todetermine the actual dose equivalent. Differ-ent types of badges are sensitive to differenttypes of radiation. Usually the Safety Officewill determine what badge is best to wearwhile working in a particular facility. Workers


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Table A.1F.1 Physical Characteristics of Commonly Used Radionuclidesa

Isotope Half-lifeDecaymode

Energy(MeV)b

Max range(cm) in

air/tissue

Dose 10 cmfrom 1 mCipoint source

Specificactivity, 100%pure (Ci/mg)

Typical use in laboratory

3Hc 12 years β- 0.0186 0.42/0 0 mrad/hr 9.6 Cell proliferation assays(3H-thymidine); taggingcellular proteins (3H-aminoacids)

14Cc 5730years

β- 0.156 22/0.027 600 mrad/hr 0.0044 Tagging cellular proteins(14C-amino acids)

32Pd 14 days β- 1.71 620/0.08 4070 mrad/hr 287 Probes for northern andSouthern blots (α-dNTPs);in vitro kinase reactions(γ-ATP); metabolic labelingof cellular proteins(ortho-32P)

33Pc 25 days β- 0.249 49/0.06 2000 mrad/hr 156 Probes for northern andSouthern blots (α-dNTPs)

35Sc 87 days β- 0.167 24/0.03 625 mrad/hr 43 Metabolic labeling ofcellular proteins or in vitrotranslation(35S-methionine/cysteine)

125Ie 60 days γ 0.027-0.035 HVL = 0.2mm lead

15 mrem/hr 17 Labeling of cell surfaceproteins or of purifiedproteins in vitro (free 125I);immunoblot detectionreagent (125I-protein A)

aTable compiled based on information in Lederer et al. (1967), Shleien (1987), and the Princeton Radiation Safety Manual (http://web.princeton.edu/

sites/ehs, see radioisotope fact sheets).bA 100 W light bulb burning for 1 hr uses 2.2 × 1018 MeV. The energy of visible light is 1.8 to 3.1 eV.cShielding is not needed for activity amounts typically used in the laboratory.dRecommended shielding for 32P is clear acrylic plastic (up to 1 cm thick for mCi amounts).eRecommended shielding for 125I is lead foil or (for mCi amounts) a leaded acrylic workstation.

should be trained to always wear their dosime-ter badge on the outside of their laboratorycoat, chest high, facing toward their work, asthe results will read low if the badge has anylayers of material (e.g., laboratory coats, jack-ets, and pocket material) covering it up. Preg-nant women are required to be educated andgiven the option to wear a dosimeter to bet-ter monitor the dose equivalent to their de-veloping fetus. When working with >1 mCiof high-energy β emitters (32P) or with anyγ/X-ray emitting isotope (125I), there is mostlikely an institutional license requirement forresearchers to wear a ring badge to measuredose to the unshielded (though gloved!) fin-gers and hands (extremities). The limit for“acceptable” exposure to the extremities is tentimes more than the limit for the whole body.However, the dose equivalent recorded withdosimeter rings may be significant compared

to the action limit for extremities set by theinstitution when working with multiple milli-curies of 32P. U.S. Federal annual dose lim-its are as follows: 5,000 mrem for the wholebody (trunk and upper extremities); 50,000mrem for the skin, extremities, and thyroid;and 15,000 mrem for the lens of the eye. Sci-entists believe that there would not be any bi-ological effect in an individual exposed to lessthan these limits during each year of their pro-fessional life (40 years used).

What is known about the risks to humansafter exposure to low levels of radiation—i.e.,levels that would be received when brieflyhandling small amounts (µCi or mCi) ofradioactivity? Unfortunately, the nature of theproblem precludes the ability of scientiststo perform controlled studies to make thisdetermination, and a complete consensus hasnot been reached. However, most experts


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think the use of a linear model extrapolateddown from the quantitatively determinedeffects of high doses of ionizing radiation isa conservative means to determine the riskper rem. These studies make use of dataobtained from studies of personnel exposuresreceived during radiation accidents, medicaltreatments, occupational exposures, and—thelargest study group—survivors of the atomicbombs dropped on Hiroshima and Nagasakiduring WWII. Genetic risks to subsequentgenerations are estimated using data fromanimal experiments and the families of atomicbomb survivors. However, since each form ofextrapolation is subject to caveats, and giventhat predictions based on such extrapolationscannot be perfect, most health and safetypersonnel aim for radiation exposure levels tobe ALARA or “as low as reasonably achiev-able.” An extensive discussion of both thestudies and statistics on which federal annualdose limits are based, which is updated on aregular basis, may be found in the BiologicalEffects of Ionizing Radiation series (BRER,2006; available online in open book form athttp://www.nap.edu/books/030909156X/html).

MINIMIZING EXPOSUREMinimizing exposure to ionizing radia-

tion can be accomplished by adjusting severalparameters of the exposure: minimizing thenumber and duration of exposures, increas-ing the distance between the researcher andthe source, and using appropriate shielding be-tween the researcher and the source.

Time is of the essenceWhen designing any experiment using ra-

dioactivity, prudent efforts should be made tolimit the time spent directly handling vials ortubes containing radioactive material or work-ing in close proximity to radioactive materials.This includes minimizing the chance of errorso as not to have to unnecessarily repeat anexperiment. Work at a pace that is comfort-able for you—not dawdling, but also not sofast as to cause spillage or error! Have every-thing needed for the experiment ready at handbefore the radioactivity is introduced into thework area. This includes materials, equipment,and a thorough knowledge of the procedurebeing performed. The more time spent famil-iarizing yourself with your protocol, the moresmoothly the work will go.

Keep your distanceWhen possible, experiments involving ra-

dioactivity should be performed in an area sep-

arate from the rest of the laboratory. Many in-stitutions require that such work be performedin a designated “hot lab”; however, if manypeople in the laboratory routinely use radioiso-topes, it is less than feasible to move them allinto what is usually a smaller space. No mat-ter where an individual is working, it is his orher responsibility to monitor the work area andensure his or her own safety and the safety ofthose working nearby. To protect bystanders,remember that the intensity of radiation froma small source (moving through air) falls offin proportion to the square of the distance.Thus, if standing 1 foot away from a source for5 min would result in an exposure of 45 mrem,standing 3 feet away for the same amount oftime would result in an exposure (1/3)2 of 45mrem, or about 5 mrem. This factor is alsorelevant when considering the storage of largeamounts of radioactivity, particularly 125I or32P, as sometimes radiation cannot be com-pletely attenuated.

Shielding: The great wallWhen handling radioactive samples, it may

be necessary to work behind shielding. Whenshielding is properly used, it will successfullyminimize researcher (and neighbor) exposureto radioactive materials. However, when usedimproperly it can lead to worker fatigue, awk-ward movements, and a higher chance ofspillage. Set up your work station in advance tomake sure you are comfortable with the physi-cal layout of your equipment and shielding. Iffeasible, start with small amounts of activity.With valuable experience and increased com-fort working with these small activity amounts,the transition to using shielding for higher ac-tivity amounts will be easier to make. Whenusing mCi amounts of 32P, shielding will al-ways be needed to minimize worker exposure.

As mentioned before, the energy of the par-ticle(s) released during the decay of an isotopedetermines what type of shielding, if any, isappropriate. β particles released during the de-cay of 14C and 35S possess roughly ten timesthe energy of those released when 3H decays.However, all three of these isotopes emit β

particles of relatively low energy, which donot travel very far in air and cannot penetratethrough solid surfaces. Therefore, these iso-topes are not considered an external hazardand shielding barriers are not necessary whenworking with them. The major health hazardfrom these isotopes is internal exposure, whichcan occur through accidental ingestion, inhala-tion, absorption through the skin, or introduc-tion through the skin by a wound.


A.1F.6


β particles released during the decay of32P may have a 10-fold higher energy thanthose released from 14C and may pose avery real concern to workers, especially ifmulti-mCi amounts of activity are being han-dled. (One potential biological effect is induc-tion of cataracts in the unshielded eye; how-ever, the threshold dose for cataracts is onthe order of 500 rem, which could only bedelivered in a laboratory setting by using mCiamounts of 32P without adequate shieldingover a span of many years.) As explainedpreviously, the fact that these high-energy β

particles can potentially generate significantamounts of bremsstrahlung radiation is the rea-son that low-Z (atomic number) materials areused as the primary layer of shielding for 32P β

radiation. Water, glass, and plastic are suitablelow-Z materials (as opposed to lead). Obvi-ously, water is unsuitable as a shielding layerfor work on the bench, although it does a rea-sonable job when samples are incubating in awater bath. Shields made from a thickness ofglass sufficient to stop these particles wouldbe extremely heavy and cumbersome (as wellas dangerous if dropped). Fortunately, plasticor acrylic materials, variously called Plexiglas,Perspex, or Lucite, are available for shieldingagainst 32P β radiation. Shields, sample stor-age boxes, waste container boxes, and sam-ple racks constructed of various thicknesses ofPlexiglas are necessary equipment in labora-tories where 32P is used. For plastic or acrylicmaterial, a thickness of about 0.64 to 1.0 cm isadequate for shielding up to 5 mCi of 32P. Toshield against bremsstrahlung radiation whenusing higher activity amounts of 32P, it is nec-essary to add a layer of high-Z material (suchas 0.38 to 2.29 mm lead) to the outside of thePlexiglas shield that is opposite the radioactivesource (Klein et al., 1990).

γ/X-rays released during the decay of 125Iwill easily penetrate the plastic materials usedto shield β particles from 32P; this radiationmust be reduced in intensity using a high-Zmaterial, such as lead. Lead foil of varyingthicknesses (0.76 to 1.0 mm) can be purchasedin rolls and can be cut and molded to cover anycontainer, or taped to a Plexiglas shield (usedin this instance for support). The latter arrange-ment has the obvious disadvantage that it is im-possible to see what one is doing through theshield. For routine shielding of manipulationsinvolving mCi activities of 125I, it is usefulto purchase a lead-impregnated, transparent,Plexiglas shield (which can be very heavy aswell as relatively expensive). When decidinghow thick is “thick enough,” consult the half-

value layer (HVL) measurement for each typeof shielding material and γ/X-ray energy. TheHVL for 125I is 0.02 mm of lead. This smallthickness of lead is sufficient to shield mostactivity amounts of 125I used in the laboratory.When using prelabeled kits, shielding is notneeded, as the activity used in these kits istypically very small (<10 µCi).

GENERAL PRECAUTIONSBefore going on to a discussion of specific

precautions to be taken with individual iso-topes, a short list of general precautions to betaken with all isotopes seems pertinent.

1. Know the rules. Be sure that each in-dividual is authorized to perform an approvedprocedure using a particular isotope and activ-ity amount in the approved work area.

2. Don the appropriate apparel. When-ever working at the laboratory bench, it isgood safety practice to wear a laboratory coatfor protection; when using radioactivity, wear-ing a laboratory coat is imperative. Dispos-able paper/synthetic coats of various stylesare commercially available: at $4 each thesemay be conveniently thrown out if contami-nated with radioactivity during an experiment,rather than held for decay as might be prefer-able with cloth coats costing ∼$30 each. Asan alternative, disposable sleeves can be pur-chased and worn over the usual cloth coat.Other necessary accessories include radiationdosimeter badges, protective eyewear, shoesthat cover the top of the foot, and two pairs ofgloves. When one of the outer gloves becomescontaminated, it is easy to slowly peel it off,replace if necessary with another glove, andcontinue working with only minor interrup-tion. Removal of contaminated gloves shouldalways be performed over a bench or wastecontainer so that microdroplets of contamina-tion do not fall on the floor and get trackedabout!

3. Protect the work area as well as theworkers. Laboratory bench tops and the basesof any shields should be covered with a dis-posable, preferably absorbent, layered papersheet. Blue absorbent pads (“hospital diapers”)work quite well.

4. Use appropriately designated equip-ment. It is very convenient, where use justifiesthe expense, to have a few adjustable pipettorsdedicated and labeled for use with each par-ticular isotope. Likewise, it is good practiceto use only certain labeled centrifuges and mi-crocentrifuge rotors for radioactive samples sothat all of the rotors in the laboratory do not


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become contaminated. Although such equip-ment should be cleaned after each use, com-plete decontamination is often not possible. Afew pipettors or a single microcentrifuge caneasily be stored (and used) behind appropriateshielding. Contamination of the insides andtip ends of pipettors can be greatly reduced byusing tips supplied with internal aerosol bar-riers such as those used for PCR reactions.To prevent contamination of the outside of thepipettor barrel, simply wrap the hand-grip inParafilm, which can be discarded later.

5. Know where to dispose of radioactivewaste, both liquid and solid. Most institu-tions require that radioactive waste be seg-regated by isotope and physical form. Thisis done not only so that appropriate shield-ing can be placed around waste containers,but so that some waste can be allowed to de-cay prior to disposal through normal (nonra-dioactive) trash methods. With a decreasingnumber of radioactive waste disposal facilitiesable or willing to accept radioactive waste forburial (and a concomitant increase in dumpingcharges from those that still do), the practiceof on-site decay can save an institution thou-sands of dollars a year in disposal charges.With this in mind, the volume of radioactivewaste present at your institution can be min-imized by surveying all items before placingthem into the radioactive waste. This will re-sult in lower costs associated with the disposalof radioactive materials.

6. Label your label! It is only commoncourtesy (as well as common sense) to alertcoworkers to the existence of anything andeverything radioactive that is left where theymay come in contact with it! A simple piece oftape affixed to the sample box with the inves-tigator’s name, the activity amount and typeof isotope, and the date written on it shouldsuffice. Yellow hazard tape printed with theinternational symbol for radioactivity is com-mercially available in a variety of widths forthis purpose.

7. Monitor for radioactive contaminationearly and often. It is imperative that each lab-oratory authorized to use radioactive materialhave access to the use of a portable radiationsurvey meter. Some meters are more suited forβ detection and some are more suited for γ

detection. Keep the appropriate survey meternearby; switch it on before touching anythingon your laboratory bench to avoid contaminat-ing the switch on the meter. Always check thebatteries before using a survey meter! Use amonitor with an adequate detector efficiency(β detector for 35S and 32P; γ detector for 125I)

before you begin, during, and after all pro-cedures. The more frequently fingers, hands,relevant equipment, and your work area aremonitored, the more quickly a spill or glovecontamination will be detected. Timely detec-tion will keep both the potential spill area andthe cleanup time to a minimum. While it istempting to cover the monitor’s detector tubewith Parafilm to protect it from contamination,remember that this will prevent the detectionof low-energy β radiation from 35S and 14C!Because the low-energy β emitter 3H cannotbe detected at all using these monitors, obtain-ing wipe samples of the bench and equipmentand subsequently counting them with a liquidscintillation counter is necessary to ensure thatcontamination of the work area did not occur.

8. Clean up contamination as soon aspossible after discovery! If contamination isdiscovered, it is to everyone’s benefit (yoursand your neighbors’) that it be cleaned up assoon as possible. This will prevent the inad-vertent spread of contamination to other areas(and people) in the laboratory. To wait over aweekend until Monday, or even one evening,to clean up contamination can invite disaster.A relatively small spill can turn into a largemess if tracked about. All things considered,it is imperative to take the time to clean up aspill immediately.

SPECIFIC PRECAUTIONSThe following sections describe precau-

tions to be taken when working with individ-ual isotopes in specific forms. Although thesections dealing with 35S- or 32P-labeling ofproteins in intact cells are presented in termsof mammalian cells, most of the instructionsare also pertinent (with minimal and obviousmodifications) to the labeling of proteins inother cells (bacterial, insect, plant, etc.).

Working with 3HTritiated compounds used in the molecular

biology laboratory include 3H-labeled thymi-dine (used in cell proliferation assays) and3H-labeled amino acids (used to label newlysynthesized proteins). As discussed above, theβ radiation resultant from the decay of tritiumis of such weak energy that no type of shield-ing is necessary to protect the scientist duringthe experiment. In fact, these β particles can-not even be detected using a typical hand-heldGeiger-Muller monitor (also known as Geigercounter or GM monitor). Therefore, to avoidaccidental ingestion or absorption through un-intentional/unrealized contact, it is imperative


A.1F.8


that the researcher perform wipe tests of the ex-perimental area and equipment used to deter-mine if any contamination exists. These wipetests most often consist of both random andspecific (most-likely candidate) swipes of sur-faces with a paper filter that is subsequentlycounted, with fluor, in a liquid scintillationcounter to determine if any 3H is/was present.

Working with 14C and 35SThese two isotopes are used to label amino

acids and thus proteins; 14C is also used to la-bel various reagents used in assays as well asmolecular weight standards to run on proteingels. As discussed above, the β radiation gen-erated during 14C or 35S decay is not strongenough to make additional shielding neces-sary. The risk associated with both these iso-topes comes primarily through their ingestionand subsequent concentration in various targetorgans, depending on the compound to whichthe radioisotope is attached. Although will-ful ingestion of either isotope seems unlikely,accidental or unknowing ingestion may occur.Additionally, the half-life of 14C is 5730 years,so do not spill this!

Using 35S to label cellular proteins andproteins translated in vitro

As reported several years ago (Meisen-helder and Hunter, 1988), 35S-labeled methio-nine and cysteine, which are routinely used tolabel proteins synthesized in intact cells andby in vitro translation, break down chemicallyto generate a volatile radioactive component.Because this breakdown occurs independentof cellular metabolism, the radioactive com-ponent is generated to the same extent instock vials as in cell culture dishes. Theprocess seems to be promoted by freezingand thawing 35S-labeled materials. The ex-act identity of this component is not known,although it is probably SO2 or CH3SH.What is known is that it dissolves readily inwater and is absorbed by activated charcoal orcopper.

The amount of the volatile radioactive com-ponent released, despite stabilizers added bythe manufacturers, is about 1/8000 of the to-tal radioactivity present. The amount of thisradioactivity that a scientist is likely to inhalewhile using these compounds is presumablyeven smaller. Nevertheless, such a componentcan potentially contaminate a wide area be-cause of its volatility, and would tend to con-centrate in target organs. Thus, it is advisableto thaw vials of 35S-labeled amino acids in a

controlled area such as a mini-hood equippedwith a charcoal filter. This charcoal filter willbecome quite contaminated and should bechanged every few months. If such an area isnot available, the stock vial should be thawedusing a needle attached to a charcoal-packedsyringe to vent and trap the volatile compound.

Anyone who has ever added 35S-labeledamino acids to dishes of cells for even shortperiods knows that the incubator(s) used forsuch labeling may quickly become highly con-taminated with 35S. Such contamination is notlimited to the dish itself, nor to the shelf onwhich the dish was placed. Rather, the radioac-tive component’s solubility in water allows itto circulate throughout the moist atmosphereof the incubator and contaminate all of the in-side surfaces of the incubator. For this reason,in laboratories where such metabolic labelingis routine, it is highly convenient to designateone incubator to be used solely for workingwith 35S-labeled samples. Such an incubatorcan be fitted with a large honeycomb-style fil-ter the size of the incubator shelf, made ofpressed, activated charcoal. These filters areavailable from local air-quality-control com-panies. Such a filter will quickly become con-taminated with radioactivity and should there-fore be monitored and changed as necessary(e.g., every three months if the incubator isused several times a week). The water usedto humidify the incubator will also becomequite “hot” (contaminated with radioactivity);keeping the water in a shallow glass pan onthe bottom of the incubator makes it easy tochange after every use, thus preventing thecontamination from accumulating. Even withthe charcoal filter and water as absorbents, theshelves, fan, and inner glass door of the incuba-tor will become contaminated, as will the trayon which the cells are carried and incubated.Routine wipe tests and cleaning when neces-sary will help to minimize potential spread ofthis contamination.

If such work is done infrequently or thereis not a “spare” incubator, dishes of cells canbe placed in a box during incubation. This boxshould be made of plastic, which is generallymore easily decontaminated than metal. Alongwith the dishes of cells, a small sachet madeof activated charcoal wrapped loosely in tis-sue (Kimwipes work well) should be placed inthe box. If the box is sealed, it will obviouslyneed to be gassed with the correct mixture ofCO2; otherwise, small holes can be incorpo-rated into the box design to allow equilibra-tion with the incubator’s atmosphere. In either


A.1F.9


Figure A.1F.1 Plexiglas shielding for 32P. (A) Two portable shields (L and T design) made of0.5-in. (12.5-mm) Plexiglas. Either can be used to directly shield the scientist from the radioactivitybeing used. Turned on its side, the L-shaped shield can be used to construct two sides of a cagearound a temporary work area, providing shielding for workers directly across from or to the sidesof the person working with 32P. (B) Tube rack for samples in microcentrifuge tubes. (C) Tube holderfor liquid waste collection.

case, the incubator used for the labeling shouldbe carefully monitored for radioactivity aftereach experiment.

Working with 32P

µCi amounts of 32PThe amount of 32P-labeled nucleotide used

to label nucleic acid probes for northern orSouthern blotting is typically under 250 µCi,and the amount of [γ−32P]ATP used forin vitro phosphorylation of proteins does notusually exceed 50 µCi for a single kinase reac-tion (or several hundred µCi per experiment).However, given the time spent on such experi-ments, handling even these small amounts canresult in a measurable dose equivalent if propershielding is not used. With no interveningshielding, the dose rate 1 cm away from 1 mCi32P is 200,000 mrads/hr; the local dose rate to

basal cells resulting from a skin contamina-tion of 1 µCi/cm2 is 9200 mrads/hr (Shleien,1987). Such a skin contamination could eas-ily be obtained through careless pipetting andthe resultant creation of an aerosol of radioac-tive microdroplets, because the concentrationof a typical stock solution of labeled nucleotidemay be 10 µCi/µl.

For proper protection during these types ofexperiments, besides the usual personal attire(glasses, gloves, coat, closed-toe shoes, andring and lapel dosimeter badges) it is nec-essary to use some form of Plexiglas shield(Fig. A.1F.1A) between the body and thesamples. Check the level of radiation com-ing through the outside of the shield witha portable monitor to ensure that the thick-ness of the Plexiglas is adequate. Hands canbe shielded from some exposure by placingthe sample tubes in a solid Plexiglas rack


A.1F.10


(Fig. A.1F.1B), which is also useful for trans-porting samples from the bench to a centrifugeor water bath.

These experiments often include an incuba-tion step performed at a specific temperature,usually in a water bath. Although the water sur-rounding the tubes or hybridization bags willeffectively stop β radiation, shielding shouldbe added over the top of the tubes where thereis no water (e.g., using a simple flat piece ofPlexiglas). If the frequency of usage justifiesthe expense, an entire lid for the water bathcan be constructed from Plexiglas. When hy-bridization reactions are performed in bags,care should be taken to monitor (and shield)the apparatus used to heat-seal the bags. It isobviously also important to ensure that the wa-ter in the bath does not become contaminatedby leakage from hybridization samples.

The waste generated during the experi-ments should also be shielded. It is convenientto have a temporary, satellite waste containerright on the bench. Pipet tips and other solidwaste can be discarded into a Plexiglas boxlined with a plastic bag and placed behindthe shield. This bag can then be emptied intothe appropriate shielded laboratory waste con-tainer when the experiment is done. Liquidwaste can be pipetted into a disposable tubeset in a stable rack or holder behind the shield(Fig. A.1F.1C).

When radiolabeled probes or proteins mustbe gel-purified, it may be necessary to shieldthe gel apparatus during electrophoresis if thesamples are particularly hot. Be advised thatthe electrophoresis buffer is likely to becomevery radioactive if the unincorporated label isallowed to run off the bottom of the gel; checkwith radiation safety personnel for instructionson how to dispose of such buffer. It is also pru-dent to check the gel plates with a radiationsurvey meter after the electrophoresis is com-pleted since they can become contaminated aswell.

mCi amounts of 32PIn order to study protein phosphorylation

in intact mammalian cells, cells in tissue cul-ture dishes are incubated in phosphate-freemedium with 32P-labeled orthophosphate fora period of several hours or overnight to labelthe proteins. The amount of 32P used in suchprocedures can be substantial. Be sure to cal-culate and pipet the actual activity, rather thanjust partitioning the label into equal volumealiquots. Cells are normally incubated in 1 to2 mCi of 32P/ml labeling medium; for a 6-cmdish of cells, 2.5 to 5 mCi 32P may be used.

When this figure is multiplied by the numberof dishes necessary per sample, and the num-ber of different samples in each experiment,the amount of 32P used in one experimentcan easily reach 25 mCi or more. Because somuch radioactivity is used in the initial label-ing phase of such experiments, it is necessaryfor a researcher to take extra precautions inorder to adequately shield him or herself andcoworkers.

When adding 32P label to dishes of cells, itis important to work as rapidly and as smoothlyas possible. An important contribution to thespeed of these manipulations is to have ev-erything that will be needed at hand beforeeven introducing the label into the work area.Prepare the work area in advance, arrangingshielding and covering the bench with blue di-apers. Set out all necessary items, includingpipettors and tips needed, a portable detec-tion monitor, extra gloves, and a cell house(Fig. A.1F.2A).

Research using this much radioactivityshould be done behind a Plexiglas shield atleast 3/4 in. (2 cm) thick; the addition of alayer of lead to the outside lower section ofthis shield to stop bremsstrahlung radiationis also needed. If one shield can be dedi-cated to this purpose at a specific location,a sheet of lead 4 to 6 mm thick can be perma-nently screwed to the Plexiglas (Fig. A.1F.2B).However, this lead makes the shield extremelyheavy and therefore less than portable. If spaceconstraints do not permit the existence of apermanent labeling station, a layer or two ofthick lead foil can be taped temporarily to theoutside of the Plexiglas shield.

Again, each worker should take care toshield not only him or herself, but also by-standers on all sides. Handling of label shouldbe done away from the central laboratory, ifpossible, to take maximum advantage of dis-tance as an additional means of dose reduction.It is also advisable not to perform such exper-iments in a tissue culture room or any otherroom that is designed for a purpose vital to thewhole laboratory. An accident involving thismuch 32P would seriously inconvenience fu-ture work in the area, if not make it altogetheruninhabitable! If care is taken to minimize theamount of time the dish of cells is open whenadding the label, use of a controlled air hoodto prevent fungal or bacterial contamination ofthe cells should not be necessary.

In the course of doing experiments to deter-mine which hand receives the most exposureduring such cell labeling procedures, extrem-ity exposure was shown to vary as much as


A.1F.11


Figure A.1F.2 (A) Box for cell incubation (a “cell house”). (B) Stationary leaded shield.(C) Sample storage rack and box made of 0.5-in. Plexiglas. (D) Box for solid waste collectionmade of 0.5-in. Plexiglas. ID, interior dimension.


A.1F.12


Figure A.1F.3 Use of Plexiglas dish shields for 32P reduces extremity exposure.

ten-fold depending on which finger thedosimeter ring was worn on, with the indexfinger of the left hand receiving the most ex-posure for a right-handed person (Bursik et al.,1999). As would be expected, the most expo-sure is received as the worker adds label tothe dishes of cells and as the cells are lysed(see below). In order to mitigate this extremityexposure, Plexiglas dish covers (Fig. A.1F.3)can be used to shield each individual dish: thetissue culture dish fits snugly into the bottomPlexiglas piece while the top Plexiglas pieceis joined to the top of the tissue culture dishusing tape so that the two lids together canbe handled as one unit. Tissue culture dishesof cells are fitted/taped into the Plexiglas dishcovers immediately before adding the 32P. Asthe top and bottom pieces of the dish coversdo not form a seal, the medium can equilibratewith the CO2 of the incubator for proper pHadjustment. Use of such dish covers reducesextremity exposure by 8- to 10-fold, despitethe stream of radiation that passes through thesmall crack between the top and bottom.

Once the label has been added to the dishesof cells (and whether or not one is using thedish covers discussed above), they will alsoneed to be shielded for transport to and fromthe incubator and other work areas. Plexi-glas boxes that are open at one end (for in-

sertion of the dishes) and have a handle ontop (for safe carrying) make ideal cell houses(Fig. A.1F.2A). A Plexiglas door that slidesinto grooves at the open end is important toprevent dishes from sliding out if the box istilted at all during transport. If this door isonly two-thirds the height of the house wall,the open slot thus created will allow equilibra-tion of the CO2 level within the house withthat in the incubator. Obviously, this slot willalso allow a substantial stream of radiation topass out of the cell house, so the house shouldbe carried and placed in the incubator with itsdoor facing away from the worker (and oth-ers)! The use of Plexiglas dish covers addsconsiderable bulk to the dishes of cells, andlarger cell houses designed with handles ontheir sides and a hinged lid are more easilyhandled (Fig. A.1F.3).

Following incubation with label and anytreatments or other experimental manipula-tions, the cells are usually lysed in some typeof detergent buffer. It is during this lysis pro-cedure that a worker’s hands will receive theirgreatest exposure to radiation, because it isnecessary to handle pipettors directly overopen cell dishes for a period of several min-utes. It is therefore very important to stream-line this procedure and use shielding when-ever possible. If the cell lysates must be made


A.1F.13


at 4C, as required by most protocols, work-ing on a bench in a cold room is preferableto placing the dishes on a slippery bed of ice.In either case, make the lysate using the samesort of shielding (with lead, if necessary) thatwas used when initially adding the label. Usingdisposable transfer pipets, remove the labelingmedium, and any solution used to rinse unin-corporated radioactivity from the cells, into asmall tube held in a solid Plexiglas holder (Fig.A.1F.1C). The contents of this tube can laterbe poured into the appropriate liquid waste re-ceptacle. If possible, it is good practice to keepthis high-specific-activity 32P liquid waste sep-arate from the lower-activity waste generatedin other procedures so that it can be removedfrom the laboratory as soon as possible fol-lowing the experiment in a specially designedshielded box. The Radiation Safety Officershould be asked to remove this high-activitywaste as soon as possible. If it is necessaryto store it in the laboratory for any time, theshielding for the waste container should alsoinclude a layer of lead.

The solid waste generated in the lysis partof these experiments (pipet tips, disposablepipets, cell scrapers, and dishes) is very hot andshould be placed immediately into a shieldedcontainer to avoid further exposure to thehands. A hinged Plexiglas box (Fig. A.1F.1D),placed to the side of the shield and lined witha plastic bag, will safely hold all radioac-tive waste during the experiment, and is lightenough to be carried easily to the main ra-dioactive waste container, where the plasticbag (and its contents) can be dumped after theexperiment is completed. If the lid of the boxprotrudes an inch or so over the front wall, itcan be lifted using the back of the hand, thusdecreasing the possibility of spreading con-tamination with hot gloves.

When scraping the cell lysates from thedishes, it is good practice to add them tomicrocentrifuge tubes that are shielded in asolid Plexiglas rack; this will help to furtherreduce the exposure to which the hands aresubjected. At this point, the lysates are usu-ally centrifuged at high speed (10,000 × g)to clear them of unsolubilized cell material.Use screw-cap tubes for this clarification step,as these will contain the labeled lysate moresecurely than flip-top tubes, which may openduring centrifugation. No matter what type oftube is used, the rotor of the centrifuge of-ten becomes contaminated, most probably be-cause tiny drops of lysate (aerosol) initiallypresent on the rim of the tubes are spun offduring centrifugation. It is important to moni-

tor and clean out the centrifuge after each useso contamination does not accumulate.

The amount of 32P taken up by cells duringthe incubation period varies considerably, de-pending on the growth state of the culture aswell as on the cell type and its sensitivity toradiation. This makes it difficult to predict thepercentage of the radioactivity initially addedto the cells that becomes incorporated into thecell lysate; however, this figure probably doesnot exceed 10%. Thus, the amount of radioac-tivity being handled decreases dramaticallyafter lysis, making effective shielding muchsimpler. Nonetheless, at least ten times moreradioactivity is still involved compared to othersorts of experiments! It is easy to determine ifthe shielding is adequate—just use both β andγ survey meters to measure the radiation pass-ing through the shielding. As a rule of thumb,if the meter reads more than 5000 cpm, addi-tional shielding is needed. Again, be sure thatpeople working nearby (including those acrossthe bench) are also adequately shielded. It issometimes necessary to construct a sort of cageof Plexiglas shields around the ice bucket thatcontains the lysates.

At the end of the day or the experiment,it may be necessary to store radioactivesamples; in some experiments, it may bedesirable to save the cell lysates. These veryhot samples are best stored in tubes placed insolid Plexiglas racks that can then be put intoPlexiglas boxes (Fig. A.1F.2C). Such boxesmay be of similar construction to the cellhouses described above, but they should havea door that completely covers the opening. Besure to check for γ radiation coming throughthese layers and add lead outside the box ifnecessary.

Working with 33P

Using 33P-labeled nucleotides to labelnucleic acid probes or proteins

Several of the major companies that manu-facture radiolabeled biological molecules alsosell nucleotides labeled with 33P (both α andγ structural forms). 33P offers a clear advan-tage over 32P with respect to ease of han-dling, because the maximum energy of theemitted β radiation is between that of 35Sand 32P and does not require as much Plex-iglas or lead thickness as is needed for 32P.In fact, the β radiation emitted (Emax=0.248MeV) can barely penetrate through two pairsof gloves and the outer dead layer of skin, sothe external exposure hazard associated witheven millicurie amounts of 33P is minimal (as


A.1F.14


reported in the DuPont NEN productbrochure). Gel bands visualized on autoradio-graphs of 33P-labeled compounds are sharperthan bands labeled with 32P because the lower-energy β radiation does not have the scatterassociated with the higher-energy β radiationof 32P. The half-life of 33P is also longer (25days compared to 14 days for 32P). Despiteits higher cost, these features have led someresearchers to choose 33P-labeled nucleotidesfor use in experiments such as band/gel shiftassays where discrimination of closely spacedgel bands is important.

The best way to determine whether addi-tional shielding is needed when using this iso-tope is to monitor the source using a β sen-sitive radiation survey meter. If counts can bedetected, add a layer of Plexiglas as describedfor 32P.

Working with 125I

Using 125I to detect immune complexes(immunoblots)

125I that is covalently attached to a moleculesuch as staphylococcal protein A is not volatileand therefore is much less hazardous than theunbound or free form. Most institutions donot insist that work with bound 125I be per-formed in a hood, but shielding of the γ ra-diation may still be necessary. Lead is a goodhigh-Z material used to shield these γ rays; itsdrawback is its opacity. Commercially avail-able shields for 125I are made of lead-loadedPlexiglas; although heavy, these have the ad-vantage of being transparent. Alternatively, apiece of lead foil may be taped to a struc-tural support, although this arrangement doesnot provide shielding for the head as a workerpeers over the lead!

Incubations of the membrane or blot withthe [125I] protein A solution and subsequentwashes are usually done on a shaker. Forshielding during these steps, a piece of leadfoil may be wrapped around the container.Solutions of 125I can be conveniently storedfor repeated use in a rack placed in a leadbox.

Using 125I to label proteins or peptidesin vitro

Any experiments that call for the use offree, unbound 125I should be done behind ashield in a hood that exhausts the air througha charcoal filter (which absorbs the volatileiodine). Most institutions require that such ex-periments be done in a special hot laboratorythat has limited access. Since ingested, ab-

sorbed, or inhaled iodine is concentrated inthe thyroid, a portable γ monitor should beused to scan the thyroid (neck area) at least24 hr after completing each experiment. Thisprocedure is called a bioassay and is a require-ment of the institution’s radioactive materialslicense. A very common means of incurring aninternal deposit of 125I during this procedureis by the spread of surface contamination withsubsequent ingestion or absorption through theskin.

MEASURING RADIOACTIVITYThere are two main purposes for count-

ing radioactive materials in the laboratory. Thefirst is to detect the presence and quantity of ra-dioactive materials in the laboratory as surfacecontamination or as an inadequately shieldedsource of radiation for the purposes of safetyand regulatory compliance. The second is todetermine the presence and/or quantity of theamount of radioactive label for the purpose ofexperimental analysis.

Safety and Regulatory ComplianceRegulations governing the use of radioac-

tive materials in the laboratory address theconcerns associated with exposure to radia-tion present as surface contamination or as anexternal radiation field (i.e., exposure at a dis-tance, typically from an inadequately shieldedradioactive source). Surface contamination issimply the unintentional presence of radioac-tive material and occurs by the transfer of ac-tual radioactive material from one surface toanother. To determine the presence of externalsurface contamination, two different method-ologies are available to the researcher: hand-held (portable) detectors and liquid scintilla-tion counters.

Hand-held detectorsThe first method is to use a portable survey

meter, i.e., either a meter with a GM detectoror a portable scintillation detector. The choicedepends on what isotope is being used. GMinstruments can be used to detect the pres-ence of the low-energy β emitters 14C, 35S,and 33P with an efficiency of detection around3% to 5%. For detecting the high-energy β

emitter 32P, the efficiency of a GM instru-ment is ∼30%. As stated previously, a layer ofParafilm, commonly used to prevent contam-ination of the GM tube in some laboratories,actually prevents the meter from detecting thelow-energy β emitters 14C, 35S, and 33P, andmust be removed prior to use.


A.1F.15


The GM detector can also be used to deter-mine the presence of 125I contamination, al-though with much lower efficiency (<1%).Because of this fact, if very small activityamounts of 125I are being used (such as pack-aged in prelabeled kits with <10 µCi), it is bestto use a portable scintillation detector, whichhas an efficiency of ∼20% for 125I.

Keep in mind that 3H β radiation has mini-mal penetration power and cannot be detectedusing either a GM or scintillation detector.The liquid scintillation counter (see below) isthe only readily available means to count 3H,either on a wipe sample or as an analyticalsample.

It is important that meter surveys be per-formed properly, as one can overlook contam-ination by either moving the meter probe toofast or by holding the probe out too far abovethe surface being monitored. This is especiallytrue for the low-energy β emitters 14C, 35S, and33P. The survey meter probe (GM or scintilla-tion detector) should be moved slowly, at about1 to 2 cm/sec, and about 1 cm above the surfaceof concern. Any response of the meter higherthan background must be considered contam-ination and cleaned up as such. The advantageof a GM survey is the greater area one cancover when doing the survey. The downsidethough is that low-level contamination can beoverlooked.

A GM detector works on the principle of theTownsend avalanche. A Townsend avalancheoccurs when a single gas molecule inside ahigh-voltage electric field becomes ionized,i.e., loses an electron by the passage of ion-izing radiation through the gas inside the GMtube. This single electron then acquires enoughkinetic energy traveling across the electric po-tential to initiate subsequent ionizations in thegas, i.e., a cascade, throughout the gas volume.This creates a large migration of free electronsto a positively charged anode, which are thenused to generate an electrical pulse in the elec-trical circuit. The avalanche in the tube is usu-ally stopped by including a quench gas in thetube, which absorbs enough of the free elec-tron energy to terminate the discharge. (Thisis a simplified explanation; there is more com-plicated physics involved that is not presentedhere.)

In a GM tube, the voltage in the tube can be600 to 2000 V and is supplied by batteries anda voltage regulator in the detector body. Theoutput of the device is a read-out on a dial, ei-ther with or without a speaker output. The GMdetector tube itself has a very thin window over

one end to allow the passage of ionizing radia-tion into the tube to initiate the avalanche. Thetube will implode if the window is accidentallypunctured because the pressure inside the tubeis lower than atmospheric pressure.

In essence, a single ionizing event inside theGM detector is amplified so it can be detected.As stated previously, the GM counter is not100% efficient at detecting all isotopes. Thisis because a threshold β energy is required topenetrate the thin window. The β energy from3H is below this energy threshold.

Liquid scintillation countersThe second method used for determining

the presence of radioactive surface contami-nation is a wipe test with subsequent count-ing in a liquid scintillation counter. A dryWhatman filter paper (no. 2 works well) isused to wipe a flat surface, using moderatepressure, in the shape of a large “S” ∼6 in.tall. The wipe is put into a liquid scintilla-tion vial, scintillation fluid (cocktail) is added,and the sample is counted in a scintillationcounter. Any counts greater than backgroundwill be indicated on the output of the counter.This method, while not in “real time,” hasthe advantage of quantifying the contamina-tion present on the wipe sample, which is noteasy to do with a GM survey meter. Exceptfor tritium, isotopes counted with this methodcan be detected with >90% efficiency. (Countscan be lost due to an effect termed quenching,which is a loss of low-energy events resultingin a shifting of the entire energy spectrum tolower energies. This results in a lower countingefficiency as compared to unquenched sam-ples.) Also, this method can detect any isotopeused in the laboratory, and can be employedto find contamination in microcentrifuges andother tight spots where a survey meter can-not fit. However, the down side is that un-less one wipes the surface by pressing directlyon the contamination itself, the contaminationwill be overlooked and a false negative canoccur.

The major advantage of a liquid scintil-lation counter over other radiation detectionequipment in the laboratory is its ability todetect and count low-energy β radiation withgood efficiency. As stated earlier, it is the onlyinstrument that can detect tritium (3H). An-other advantage is its ability to discriminatebetween high- and low-energy β radiation.Indeed, samples that contain more than oneisotope can be counted and the proportion ofeach separate isotope determined. The device


A.1F.16


is quite complex, and its operation dependson many principles of electronics, electrome-chanics, and physics.

Vital to liquid scintillation counting is theuse of a scintillation fluid. A scintillationfluid is a specially formulated liquid com-prised of two kinds of molecules: scintilla-tion molecules and, in much greater quantity,solvent molecules. Together these moleculesserve the purpose of transforming the energyof the emitted radiation inside a sample to anenergy level that falls in the sensitivity rangeof a photomultiplier tube (PMT). The radia-tion energy in the sample is first transferred tothe solvent, which has an energy level abovethat of the scintillant and the PMT. The en-ergy is then transferred to the scintillant, whichis stimulated to a higher energy level. As thescintillant returns to its ground state, it releasesenergy or light that lies within the sensitivityof the PMT. The light enters the PMT and initi-ates a cascade down a string of dynodes, wherethe PMT converts a relatively small light sig-nal at one end into a large light pulse at theother. This pulse is then processed electricallyand counted.

The fact that the sample is in intimate con-tact with the fluid, usually dissolved or insolution, accounts for the greater sensitivityof this device over that of the GM detector.The important fact is that higher β energiesin the sample will stimulate greater numbersof solvent molecules in the first place, lead-ing to a larger number of stimulated scintillantmolecules. This in turn results in a more in-tense event at the start of the dynode stringand a greater pulse at the other end. This ex-plains how these detectors can discriminateβ radiation of different energies. In terms ofliquid scintillation cocktails (fluors), keep inmind that some chemicals, notably solventshistorically used in liquid scintillation cocktailformulations, are very difficult and expensiveto dispose of as radioactive waste, as someare classified as “mixed wastes.” It is best totry to avoid these solvents. Some institutionshave restrictions on their use, so the RadiationSafety Officer should be contacted for moredirection. The best cocktails to use are envi-ronmentally friendly cocktails.

External radiation fields: Exposure at adistance

Radiation present in the form of an externalradiation field (exposure at a distance) is alsopossible in the laboratory. This occurs mostoften when inadequately shielded mCi activ-ities of 32P are present. In this scenario, high

local dose rates are possible at near distancesand, because of the long range of 32P β radi-ation in air, dose rates even a few feet fromthe source can be substantial. This radiationfield cannot be measured directly with a GMtype of survey instrument, although a roughestimate of the dose rate can be made. Usingthe very common, large area “pancake” typeof GM probe, a meter response to 32P of 2000to 3000 cpm indicates an approximate doserate of 1 mrem per hour. This is a good levelto aim for when determining the adequacy ofany added shielding used to minimize the doserate from mCi 32P sources. Again, this is only arough estimate, and Radiation Safety Depart-ment personnel should be contacted to pro-vide more accurate determinations of exter-nal dose rates. Ion chamber type instrumentsare available that can measure actual dose orexposure rates more closely than GM surveyinstruments.

Obviously, the best way to perform a com-plete laboratory survey is to use a combina-tion of the two types of surveys (direct metersurvey and wipe test survey) in such a wayas to increase the probability of finding sur-face contamination and/or any inadequatelyshielded radioactive sources. One can neverdo too many surveys, and the more experi-ence one gains, the more successful one willbecome at locating and cleaning radioactivecontamination in the laboratory.

Experimental AnalysisDue to technological advances with imag-

ing machines and computer software, there isnow an increasing number of ways to deter-mine the amount of radioactivity present inexperimental samples. However, if one wantsto know the actual activity (dpm or cpm) of anisotope present, the most straightforward wayis still to use a scintillation counter to count thematerial. While this is often the final stage ofan experiment, in that the samples have beenprepared with this quantitation being the goal,sometimes this measurement is necessary atseveral points in an experiment to monitorrecovery of the sample. Since low-energy β

emitters (3H, 14C, or 35S) require the use of ascintillation fluor, the portion of sample thatis counted is lost in terms of further analysis.While liquid scintillation counting is more ac-curate, 32P samples offer the option of usingCerenkov counting, which preserves the entiresample for future types of analysis. Cerenkovcounting requires a β Emax of at least 0.7 MeV,so 32P samples can be counted with goodefficiency using this method. For Cerenkov


A.1F.17


counting, the sample is simply put into anempty scintillation vial and counted; thecounts appearing in the wide window (32H plus14C plus 32P) are proportional to the amountof 32P present. Keep in mind that Cerenkovcounting of a dry sample (a gel band or pro-tein pellet, for example) cannot be directlycompared with that of a sample counted withliquid scintillation fluid because the liquid inthe sample will act to quench some of the ra-dioactivity. For 125I samples, a γ scintillationcounter is used; samples can be dry or in liq-uid form, and no fluor is necessary. Liquidscintillation can also be used to count 125Isamples.

The advent of phosphor screen technologyhas enabled researchers to both image andquantitate gels, blots, or plates by exposingthem to a screen and then enabling the in-strument to read the screen. The screens candetect 35S, 32P, and 125I with roughly 5-to 10-fold higher sensitivity than film (depending onthe isotope) and with a detection range of sam-ple variability over 5 orders of magnitude (asopposed to 2 orders for film). Computer soft-ware is used to select areas of the resultant im-age and “extract” the amount of radioactivitytherein. The numbers thus generated are of ar-bitrary units, but can be used for comparativepurposes, the caveats being that the extractedareas must be from the same screen, whichitself must be undamaged so that its sensi-tivity is uniform. If standards of known ra-dioactive content are used to expose the screenalongside the sample, the arbitrary units gen-erated can be translated into real cpm. Severalcompanies now make such instruments, withFuji, BioRad, and Molecular Dynamics (nowAmersham) being among the best known. Thecost of these instruments is still very high; forthis reason many institutions purchase one foruse in a core facility. The ability to “cut andcount” on a computer rather than on real sam-ples is highly convenient, and the larger rangeof sensitivity of the phosphor screen eliminatesthe need for multiple exposures, as would benecessary when using film and a densitometerto quantify the signal.

RESPONDING TO SPILLSDespite the best intentions and utmost cau-

tion, accidents happen! Accidents involvingspills of radioactive materials are particularlyinsidious because they can be virtually unde-tectable if a monitor is not present and turnedon. For this reason it is best to foster a com-munity spirit in any laboratory where radioiso-topes are routinely used—specifically, a sense

of cooperation that extends from shieldingwith others in mind to helping each other cleanup after accidents occur.

The specific measures to be taken follow-ing a spill of radioactive materials naturallydepend on the type and activity amount of iso-tope involved, the associated chemical or bio-logical hazards, and the physical parameters ofthe spill (i.e., where and onto what the isotopewas spilled). However, there are several im-mediate steps that should be taken followingany spill.

1. Alert coworkers that there has beena spill. This will give them the opportunityto protect themselves if need be and to helpprepare to clean up the spill. Depending onthe size and nature of the spill, it will oftenbe necessary to notify the Radiation SafetyOffice.

2. Restrict movement to and from the siteof the spill. This ensures that radioactive con-tamination is not spread around the laboratory.It is especially important to address those indi-viduals who may have come into contact withthe radioactive materials.

3. Perform a meter survey on anyindividuals who may have come in contactwith the radioactive material, including theirexposed skin, protective clothing, and streetclothing. If someone’s skin is contaminated,first use a portable monitor to identify the spe-cific areas of contamination. Then put that partof the body under room temperature runningwater in a sink. Wash the affected area withgentle soap and a soft sponge or washcloth.Try to restrict cleaning to the contaminatedarea only, so as not to spread the contamina-tion to other parts of the body. Dry the area,survey, and repeat as necessary or as directedby Radiation Safety personnel. A shower roommay be needed. Contaminated clothing can becarefully removed, placed in a plastic bag, andgiven to Radiation Safety personnel. Contam-inated strands of hair can be washed or cut(a new hairstyle may be in order).

4. Perform an area survey. Use an ap-propriate survey meter and work in from thesupposed outer limits of the spill towards thecenter. With a nonpermanent marker, outlinethe actual hot spots where counts are detected.Do not neglect to survey the sides of walls,cabinets, and equipment close to the spill area,as radioactive materials may have splashed uponto these surfaces.

5. Clean the area. When attempting toclean any contaminated equipment, floors,benches, etc., begin by soaking up any visi-ble radioactive liquid with paper towels and


A.1F.18


promptly disposing of the towels in a plasticbag taped to the side of the laboratory bench.Apply a small amount of decontaminationsolution to the marked “hot spots” and let itset for a few minutes. Then, using three tofour paper towels, wipe an area from the outeredge to the center of the spill with one swipeof the towels. Immediately discard the papertowels. Repeat this movement with new papertowels each time, working your way aroundthe spill. Do not reuse any paper towels. Thismethod will minimize the chance of spreadingcontamination to an even greater area. Con-tinue this procedure until all the marked areashave been cleaned.

6. Perform another radiation survey.Survey the entire area to make sure contam-ination has not been overlooked.

There is quite a range of commerciallyavailable foams and sprays made specificallyto clean radioactive contamination. A dilutesolution of phosphoric acid works well to pickup 32P. Decontamination of centrifuge rotorscan be tricky as their anodized surfaces aresensitive to many detergents; check with therotor manufacturer for appropriate cleansingsolutions. Many surfaces (particularly metals)prove resistant to even herculean cleaning ef-forts; in these instances the best that can bedone is to remove all contamination possi-ble and then shield whatever remains until theradioactivity decays to minimal levels.

CONCLUSIONWhen working with radioisotopes, it is best

to plan ahead and then plan ahead some more.The more thoroughly familiar a researcher be-comes with all aspects of their work—by be-ing proactive and addressing all questions andconcerns beforehand (the science, the equip-ment, and the technique)—the more success-ful the researcher will be at providing for theirown safety and the safety of those workingaround them.

ACKNOWLEDGEMENTSMany of the procedures and precautions de-

scribed here have evolved over the years andthrough the millicuries (and are evolving still)in the authors’ department (currently Molecu-lar and Cell Biology) at the Salk Institute. Theauthors are indebted to those from whom theyhave learned about the safe use of radioactiv-ity in the laboratory. Most of the designs forthe shields and other safety equipment shownin the figures were created at the Salk Insti-tute in collaboration with Dave Clarkin, MarioTengco, and Steve Berry. Safety equipment ofsimilar design is available from several com-mercial vendors, including CBS Scientific andResearch Products International.

LITERATURE CITEDBoard on Radiation Effects Research (BRER).

2006. Health risks from exposure to lowlevels of ionizing radiation: BEIR VIIPhase 2. BRER, National Research Council,The National Academies Press, Washington,D.C. Available online at http://www.nap.edu/books/030909156X/html.

Bursik, S., Meisenhelder J., and Spahn, G. 1999.Characterization and minimization of extremitydoses during 32P metabolic cell labeling. HealthPhys. 77:595-600.

Klein, R., Reginatto, M., Party, E., and Gershey, E.1990. Practical radiation shielding for biomedi-cal research. Radiat. Prot. Manage. 7:30-37.

Lederer, C.M., Hollander, J.M., and Perlman, I.(eds.) 1967. Table of Radioisotopes, 6th ed. JohnWiley & Sons, New York.

Meisenhelder, J. and Hunter, T. 1988. Radioactiveprotein-labeling techniques. Nature 335:120.

Shleien, B. (ed.) 1987. Radiation Safety Manual forUsers of Radioisotopes in Research and Aca-demic Institutions. Nucleon Lectern Associates,Olney, Md.

INTERNET RESOURCEShttp://web.princeton.edu/sites/ehsPrinceton University Environmental Health andSafety Web site containing radioisotope fact sheets.

APPENDIX 1GCentrifuges and Rotors

Centrifugation runs described in this book usually specify a relative centrifugal force(RCF; measured in × g), corresponding to a speed (in rpm) for a particular centrifuge androtor model. As available equipment will vary from laboratory to laboratory, the investi-gator must be able to adapt these specifications to other centrifuges and rotors.

The relationship between RCF and speed (rpm) is determined by the following equation:

RCF =1.12r (rpm ⁄ 1000)2

where r is the rotating radius between the particle being centrifuged and the axis ofrotation. In most cases, an accurate conversion from speed to relative centrifugal force(or vice versa) can be obtained using the maximum value of r—or rmax—equal to thedistance between the axis of rotation and the bottom of the centrifuge tube as it sits in thewell or bucket of the rotor.

Table A.1G.1 provides rmax values for commonly used rotors manufactured by Du Pont(Sorvall), Beckman, Fisher, and IEC. There are situations (e.g., where an adapter is usedto fit a smaller tube into a larger rotor well) where rmax will not accurately represent theeffective rotating radius. In such cases, the manual for the rotor should be consulted toobtain the appropriate value of r.

As an alternative to use of the above equation, the nomograms in Figures A.1G.1 andA.1G.2 make it possible to determine the RCF where speed and rmax are known, or thespeed where RCF and rmax are known. This is done by aligning a ruler across the twoknown values and reading the unknown value at the point where the ruler crosses theremaining column. Figure A.1G.1 should be used for centrifuge runs <21,000 rpm, whileFigure A.1G.2 should be used for faster spins.

NOTE: In this manual, for spins involving microcentrifuges built to the Eppendorfstandard, a shortened style of reference including only the speed (in rpm) is used. All ofthese instruments have approximately the same rotating radius; hence the same speed willyield the same RCF value from machine to machine. Microcentrifuge spins may also bedescribed as at “top speed” or “maximum speed,” meaning 12,000 to 14,000 rpm, whichis the maximum speed for all Eppendorf-type microcentrifuges.

CAUTION: Do not exceed maximum rotor speed! For Beckman ultracentrifuges, themaximum speed for each rotor is denoted by its name, e.g., the maximum speed of theBeckman VTi 80 rotor is 80,000 rpm. This speed refers only to centrifugation of solutionsbelow a particular allowed density, which differs among rotors (see user manual). Forcentrifugation of high-density solutions, rotor maximum speed can be determined as:reduced rpm = rpmmax(A/B)

1⁄2, where A=allowed density and B=density of solution. A=1.7g/ml for several vertical rotors (including VTi 80 and VTi 50), and 1.2 g/ml for severalswinging-bucket rotors (including SW 55 Ti, 28, 28.1, 40 Ti, 50.1). For gradients usingheavy salts such as CsCl, particularly at low temperatures, maximum rpm should bereduced to prevent precipitation (see user manual).

Table A.1G.2 describes centrifuge tube materials and their properties, including opticalproperties, appropriate methods for sterilization, and chemical resistances (tolerance tovarious media, organic solvents, and alcohols).

Supplement 35

Current Protocols in Molecular Biology (1996) A.1G.1-A.1G.6Copyright © 2000 by John Wiley & Sons, Inc.

A.1G.1


90,000

50,000

10,000

5000

1000

500

200

100

5040

30

20

15.7

Relative centrifugalforce (x g)

180

150

100

90

80

70

60

50

30

40

25 750

1000

2000

5000

10,000

15,000

20,00021,000

Rotating rad ius(mm)

Rotor speed(rpm)

Figure A.1G.1 Nomogram for conversion of relative centrifugal force to rotor speed in low-speed centrifuge runs. Todetermine an unknown value in a given column, align ruler through known values in other two columns. Desired valueis found at the intersection of the ruler with the column of interest. For faster centrifugations, use Figure A.1G.2. Amore precise conversion can be obtained using the equation at the beginning of this appendix. See Table A.1G.1 forrotating radii of commonly used rotors.


A.1G.2

Centrifugesand Rotors

200

180

160

140

120

100

90

70

60

80

50

40

30

20

10

1000

2000

3000

4000

50006000700080009000

10,000

20,000

30,000

40,000

50,000

60,00070,00080,00090,000

100,000

200,000

300,000

400,000

500,000600,000700,000800,000900,000

1,000,000

80,00075,000

70,000

65,000

60,000

50,000

40,000

30,000

20,000

10,000

Rotating radius(mm)

Relative centrifugalforce (x g )

Rotor speed(rpm)

Figure A.1G.2 Nomogram for conversion of relative centrifugal force to rotor speed in high-speedcentrifuge runs. For slower centrifugations and instructions for using the nomogram, use Figure A.1G.1.A more precise conversion can be obtained using the equation at the beginning of this appendix. SeeTable A.1G.1 for rotating radii of commonly used rotors.


A.1G.3

Table A.1G.1 Maximum Rotating Radii for Common Rotors, Grouped by Centrifuge Model

Rotor modela rmax (mm) Rotor modela rmax (mm)

For Sorvall centrifuge models GLC-1,GLC-2, GLC-2B, GLC-3, GLC-4, RT-6000B, T-6000, T-6000BA/S400 140H-1000B 186HL-4 with 50-ml bucket 180HL-4 with 100-ml bucket 204HL-4 with Omni-Carrier 163M and A-384 (inner row) 91M and A-384 (outer row) 121SP/X and A-500 (inner row) 82SP/X and A-500 (outer row) 123

For Sorvall centrifuge models RC-3, RC-3B, RC-3C)H-2000B 261H-4000 and HG-4L 230H-6000A 260HL-8 with Omni-Carrier 221HL-8 with 50-ml bucket 238HL-8 with 100-ml bucket 247HL-2 and HL-2B 166LA/S400 140

For Sorvall centrifuge models RC-2, RC-2B, RC-5, RC-5B, RC-5CGSA 145GS-3 151HB-4 147HS-4 with 250-ml bucket 172SA-600 129SE-12 93SH-80 101SM-24 (inner row) 91SM-24 (outer row) 110SS-34 107SV-80 101SV-288 90TZ-28 95

For Sorvall ultracentrifugesT-865 91T-865.1 87.1T-875 87.1T-880 84.7T-1270 82TFT-80.2 65.5TFT-80.4 60.1For Beckman GP series centrifugesGA-10 123GA-24 123GA-24 with adapter for 10-ml tubes

108

GH-3.7 (buckets) 204GH-3.7 (microplate carrier) 168GH-3.8 (buckets) 204GH-3.8 (microplate carrier) 168

For Beckman TJ-6 series centrifugesTA-10 123TA-24 108TA-24 with adapter for 10-ml tubes

123

TH-4 (stainless steel buckets)

186

TH-4 (100-ml tube holders) 201TH-4 (microplate carrier) 165

For Beckman AccuSpinAA-10 123AA-24 108AA-24 with adapter for 10-ml tubes

123

AH-4 163

For Beckman J6 series centrifugesJR-3.2 206JS-2.9 265JS-3.0 254JS-4.0 226JS-4.2 254JS-4.2SM 248JS-5.2 226Microplate carrier (6-bucket rotors)

214

Microplate carrier (4-bucket rotors)

192

For Beckman J2-21 series centrifugesJA-10 158JA-14 137JA-17 123JA-18 132JA-18.1 (25° angle) 112

JA-18.1 (45° angle) 116JA-20 108JA-20.1 115JA-21 102JCF-Z 89JCF-Z with small pellet core 81JE-6B 125JS-7.5 165JS-13 142JS-13.1 140JV-20 93

continued


A.1G.4

Centrifugesand Rotors

Table A.1G.1 Maximum Rotating Radii for Common Rotors, Grouped by Centrifuge Model, continued

Rotor modela rmax (mm) Rotor modela rmax (mm)

For Beckman series L7 and L8ultracentrifugesSW 25.1 129.2SW 28 161.0SW 28.1 171.3SW 30 123.0SW 30.1 123.0SW 40 Ti 158.8SW 41 Ti 153.1SW 50.1 107.3SW 55 Ti 108.5SW 60 Ti 120.3SW 65 Ti 89.0Type 15 142.1Type 19 133.4Type 21 121.5Type 25 100.4Type 30 104.8Type 30.2 94.2Type 35 104.0Type 40 80.8Type 40.3 79.5Type 42.1 98.6Type 42.2 Ti 104Type 45 Ti 103Type 50 70.1Type 50 Ti 80.8Type 50.2 Ti 107.9Type 50.3 Ti 79.5Type 50.4 Ti (inner row) 96.4Type 50.4 Ti (outer row) 111.4Type 55.2 Ti 100.3Type 60 Ti 89.9Type 65 77.7Type 70 Ti 91.9Type 70.1 Ti 82.0Type 75 Ti 79.7

Type 80 Ti 84.0VAC 50 86.4VC 53 78.8VTi 50 86.6VTi 65 85.4VTi 65.2 87.9VTi 80 71.1

For Beckman Airfuge ultracentrifugeA-95 17.6A-100/18 14.6A-100/30 16.5A-110 14.7ACR-90 (2.4-ml liner) 11.8ACR-90 (3.5-ml liner) 13.4Batch rotor 14.6EM-90 13.0

For Beckman TL-100 series ultracentrifugesTLA-100 38.9TLA 100.1 38.9TLA-100.2 38.9TLA-100.3 48.3TLA-45 55.1TLS-55 76.4TLV-100 35.7

Miscellaneous centrifuges and rotorsb

Clay Adams Dynac —c

Fisher Centrific 113Fisher Marathon 21K with 4-place rotor

160

IEC Clinical centrifuge with 4-place swinging-bucket rotor

155

IEC general-purpose centifuge models HN, HN-SII, and Centra-4

—c

aSorvall centrifuges and rotors are a product of Du Pont Company Medical Products, Beckman centrifuges are a productof Beckman Instruments, IEC centrifuges are a product of International Equipment Co., Clay Adams Dynac centrifugesare a product of Becton Dickinson Labware, and Fisher centrifuges are a product of Fisher Scientific. For orderinginformation see APPENDIX 4.bThese instruments are often loosely referred to as “clinical,” “tabletop,” or “low-speed” centrifuges.cThese instruments accept a wide range of trunnion-ring rotors with variable rotating radii, as well as fixed-angle andswinging-bucket rotors that in turn accept a variety of adapters making it possible to spin different numbers tubes ofvarious sizes. For instance, the commonly used IEC 958 trunnion-ring rotor may be adjusted to a radii ranging from 137to 181 mm, depending on the trunnion-ring chosen. It is therefore necessary to consult the manual for the specific systembeing used to obtain an accurate speed to RCF conversion.


A.1G.5


Tab

le A

.1G

.2

Cen

trifu

ge T

ube

Mat

eria

ls a

nd T

heir

Pro

pert

iesa

Type

Opt

ical

prop

erty

Punc

tura

ble

Slic

eabl

eR

eusa

ble

Ster

iliza

tion

met

hods

Che

mic

al r

esis

tanc

esb

Ultr

a-C

lear

thin

-wal

led

Tra

nspa

rent

Y

es

Yes

Col

d st

erili

zatio

n on

ly, b

ut n

otw

ith a

lcoh

olG

ood

tole

ranc

e to

all

grad

ient

med

ia e

xcep

tal

kalin

e on

es (

>pH

8).

Sat

isfa

ctor

y fo

r m

ost w

eak

acid

s an

d a

few

wea

k ba

ses.

Uns

atis

fact

ory

for

DM

SO

and

mos

t org

anic

sol

vent

s, in

clud

ing

all

alco

hols

.

St

anda

rd tu

bes

Y

es

Qui

ck-S

eal t

ubes

N

o

Poly

allo

mer

thin

-wal

led

Tra

nslu

cent

Y

es

Yes

Can

be

auto

clav

ed o

n a

test

tube

rack

at 1

21°C

Goo

d to

lera

nce

to a

ll gr

adie

nt m

edia

, inc

ludi

ngal

kalin

e on

es. S

atis

fact

ory

for

mos

t aci

ds, m

any

base

s, m

any

alco

hols

, DM

SO, a

nd s

ome

orga

nic

solv

ents

.

St

anda

rd tu

bes

Y

es

Qui

ck-S

eal t

ubes

N

o

Poly

allo

mer

thic

k-w

alle

dT

rans

luce

ntC

an b

e au

tocl

aved

on

a te

st tu

bera

ck a

t 121

°CG

ood

tole

ranc

e to

all

grad

ient

med

ia, i

nclu

ding

alka

line

ones

. Sat

isfa

ctor

y fo

r m

ost a

cids

, man

yba

ses,

man

y al

coho

ls, D

MSO

, and

som

e or

gani

cso

lven

ts.

T

ubes

N

o

No

Y

es

Bot

tles

N

o

No

Y

es

Poly

carb

onat

e th

ick-

wal

led

Tra

nspa

rent

N

o

No

Col

d st

erili

zatio

n re

com

men

ded,

but n

ot w

ith a

lcoh

ol. C

an b

eau

tocl

aved

at 1

21°C

, but

tube

life

may

be

redu

ced.

Goo

d to

lera

nce

to a

ll gr

adie

nt m

edia

exc

ept

alka

line

ones

(>p

H 8

). S

atis

fact

ory

for

som

e w

eak

acid

s. U

nsat

isfa

ctor

y fo

r al

l bas

es, a

lcoh

ols,

and

othe

r or

gani

c so

lven

ts.

T

ubes

Y

es

Bot

tles

Y

es

Cel

lulo

se p

ropi

onat

e tu

bes

Tra

nspa

rent

N

o

No

Y

esC

old

ster

iliza

tion

only

, but

not

with

alc

ohol

Goo

d to

lera

nce

to a

ll gr

adie

nt m

edia

, inc

ludi

ngal

kalin

e on

es. U

nsat

isfa

ctor

y fo

r m

ost a

cids

,ba

ses,

alc

ohol

s, a

nd o

ther

org

anic

sol

vent

s.

Poly

prop

ylen

eT

rans

luce

nt

No

N

oC

an b

e au

tocl

aved

at 1

21°C

Goo

d to

lera

nce

to a

ll gr

adie

nt m

edia

, inc

ludi

ngal

kalin

e on

es. S

atis

fact

ory

for

man

y ac

ids,

bas

es,

and

alco

hols

. Uns

atis

fact

ory

for

mos

t org

anic

solv

ents

.

T

ubes

Y

es

Bot

tles

Y

es

Stai

nles

s st

eel t

ubes

Opa

que

N

o

No

Y

esC

an b

e au

tocl

aved

. Dry

thor

ough

ly b

efor

e st

orag

e.G

ood

tole

ranc

e to

man

y or

gani

c so

lven

ts.

Mar

gina

l with

man

y gr

adie

nt m

edia

and

sal

ts.

Uns

atis

fact

ory

for

mos

t aci

ds a

nd m

any

base

s.Po

lyet

hyle

ne tu

bes

Tra

nslu

cent

N

o

No

Y

esC

an b

e au

tocl

aved

at 1

21°C

Goo

d to

lera

nce

to a

wid

e ra

nge

of c

hem

ical

s.Su

itabl

e fo

r us

e w

ith s

tron

g ac

ids

and

base

s.U

nsat

isfa

ctor

y fo

r m

ost o

rgan

ic s

olve

nts.

Cor

ex/P

yrex

Tra

nspa

rent

N

o

No

Can

be

auto

clav

ed a

t 121

°CG

ood

tole

ranc

e to

a w

ide

rang

e of

gra

dien

t med

ia.

Cor

ex h

as g

reat

er r

esis

tanc

e to

bas

es a

nd a

cids

.

Tub

es

Yes

B

ottle

s

Yes

a Tabl

e re

prod

uced

by

perm

issi

on o

f B

eckm

an I

nstr

umen

ts.

b Che

mic

al r

esis

tanc

es a

re d

escr

ibed

her

e in

gen

eral

ter

ms,

and

are

not

mea

nt b

y B

eckm

an I

nstr

umen

ts o

r Jo

hn W

iley

& S

ons

to e

xpre

ss o

r im

ply

any

guar

ante

e of

saf

ety

base

d on

the

sere

com

men

datio

ns o

r res

ista

nces

. If t

here

is a

ny d

oubt

abo

ut a

par

ticul

ar s

olut

ion,

it s

houl

d be

test

ed u

nder

act

ual o

pera

ting

cond

ition

s to

eva

luat

e th

e pe

rfor

man

ce o

f a tu

be m

ater

ial.

For m

ore

deta

iled

info

rmat

ion

rega

rdin

g sp

ecif

ic m

edia

and

sol

vent

s, c

onsu

lt B

eckm

an I

nstr

umen

ts. H

igh-

vapo

r-pr

essu

re in

flam

mab

le s

olve

nts

shou

ld n

ot b

e ha

ndle

d in

clo

se v

icin

ity to

cen

trif

uges

beca

use

of p

ossi

ble

igni

tion

by s

park

ing

switc

hes,

rel

ay c

onta

cts,

or

mot

or b

rush

es.

nnnn

A.1G.6


APPENDIX 1HSafe Use of Hazardous ChemicalsCarrying out the protocols in this manual may result in exposure to toxic chemicals orcarcinogenic, mutagenic, or teratogenic reagents (see Table A.1H.1). Cautionary notesand some specific guidelines are included in many instances throughout the manual;however, users must proceed with the prudence and precautions associated with goodlaboratory practice, under the supervision of those responsible for implementing labsafety programs at their institutions.

It is not possible in the space available to list all the precautions required for handlinghazardous chemicals. Many texts have been written about laboratory safety (see LiteratureCited and Key References). Obviously, all national and local laws should be obeyed, aswell as all institutional regulations. Controlled substances are regulated by the DrugEnforcement Administration (http://www.doj.gov/dea). By law, Material Safety DataSheets (MSDSs) must be readily available. All laboratories should have a ChemicalHygiene Plan (29CFR Part 1910.1450); institutional safety officers should be consultedas to its implementation. Help is (or should be) available from your institutional SafetyOffice; use it.

Chemicals must be stored properly for safety. Certain chemicals cannot be easily or safelymixed with and should not be stored near certain other chemicals, because their reactionis violently exothermic or yields a toxic product. Some examples of incompatibility arelisted in Table A.1H.2. When in doubt, always consult a current MSDS for informationon reactivity, handling, and storage. Chemicals should be separated into general hazardclasses and stored appropriately. For example, flammable chemicals such as alcohols,ketones, aliphatic and aromatic hydrocarbons, and other materials labeled flammableshould be stored in approved flammable storage cabinets, with those also requiringrefrigeration being kept in explosion-proof refrigerators. Strong oxidizers must be segre-gated. Strong acids (e.g., sulfuric, hydrochloric, nitric, perchloric, and hydrofluoric)should be stored in a separate cabinet well removed from strong bases and from flammableorganics. An exception is glacial acetic acid, which is both corrosive and flammable, andwhich must be stored with the flammables.

Facilities should be appropriate for working with hazardous chemicals. In particular,hazardous chemicals should be handled only in chemical fume hoods, not in laminar flowcabinets. The functioning of the fume hoods should be checked periodically. Laboratoriesshould also be equipped with safety showers and eye-wash facilities. Again, this equip-ment should be tested periodically to ensure that it functions correctly. Other safetyequipment may be required depending on the nature of the materials being handled. Inaddition, researchers should be trained in the proper procedures for handling hazardouschemicals as well as other laboratory operations—e.g., handling of compressed gases,use of cryogenic liquids, operation of high-voltage power supplies, and operation of lasersof all types.

Before starting work, know the physical and chemical hazards of the reagents used. Wearappropriate protective clothing and have a plan for dealing with spills or accidents; comingup with a good plan on the spur of the moment is very difficult. For example, have theappropriate decontaminating or neutralizing agents prepared and close at hand. Smallspills can probably be cleaned up by the researcher. In the case of larger spills, the areashould be evacuated and help should be sought from those experienced in and equippedfor dealing with spills—e.g., the institutional Safety Office.

Protective equipment should include, at a minimum, eye protection, a lab coat, and gloves.In certain circumstances other items of protective equipment may be necessary (e.g., aface shield). Different types of gloves exhibit different resistance properties (Table

Supplement 58

Contributed by George Lunn and Gretchen LawlerCurrent Protocols in Molecular Biology (2002) A.1H.1-A.1H.33Copyright © 2002 by John Wiley & Sons, Inc.

A.1H.1


Table A.1H.1 Commonly Used Hazardous Chemicalsa

Chemical Hazards Remarksb

Acetic acid, glacial Corrosive, flammable liquid

Acetonitrile Flammable liquid, teratogenic, toxic

Acridine orange Carcinogenic, mutagenic See Basic Protocol 2

Acrylamide Carcinogenic, toxic Use dust mask;polyacrylamide gels containresidual acrylamidemonomer and should behandled with gloves;acrylamide may polymerizewith violence on melting at86°C

Alcian blue 8GX See Basic Protocol 2

Alizarin red S (monohydrate)

p-Amidinophenylmethanesulfonyl fluoride (APMSF)

Enzyme inhibitor See Basic Protocol 11

7-Aminoactinomycin D (7-AAD) Carcinogenic

4-(2-Aminoethyl)benzenesulfonyl fluoride (AEBSF)

Mutagenic, enzyme inhibitor See Basic Protocol 11

Ammonium hydroxide, concentrated Corrosive, lachrymatory, toxic

Azure A Mutagenic See Basic Protocol 2

Azure B Mutagenic See Basic Protocol 2

Benzidine (BDB) Carcinogenic, toxic See Basic Protocol 1

Bisacrylamide Toxic

Boron dipyrromethane derivatives (BODIPY dyes)

Toxic

Brilliant blue R Carcinogenic, mutagenic See Basic Protocol 2

5-Bromodeoxyuridine (BrdU) Mutagenic, teratogenic,photosensitizing

Cetylpyridinium chloride (CPC) Toxic

Cetyltrimethylammonium bromide (CTAB) Corrosive, teratogenic, toxic

Chloroform Carcinogenic, teratogenic, toxic

Chlorotrimethylsilane Carcinogenic, corrosive, flammableliquid, toxic

Reacts violently with water;see Basic Protocol 3

Chromic/sulfuric acid cleaning solution Carcinogenic, corrosive, oxidizer,toxic

Replace with suitablecommercially availablecleanser

Chromomycin A3 (CA3) Teratogenic, toxic

Congo red Mutagenic, teratogenic See Basic Protocol 2

Coomassie brilliant blue G Mutagenic See Basic Protocol 2

Crystal violet See Basic Protocol 2

Cresyl violet acetate Mutagenic See Basic Protocol 2

Cyanides (e.g., KCN, NaCN) Toxic Contact with acid willliberate HCN gas; see BasicProtocol 4

Cyanines (e.g., Cy3, Cy5) Toxic

Cyanogen bromide (CNBr) Toxic See Basic Protocol 4

continued


A.1H.2

2′-Deoxycoformycin (dCF, pentostatin) Teratogenic, toxic

4′,6-Diamidino-2-phenylindole (DAPI) Mutagenic

Diaminobenzidine (DAB) Carcinogenic See Basic Protocol 1

1,4-Diazabicyclo[2,2,2]-octane (DABCO) Toxic Forms an explosive complexwith hydrogen peroxide

Dichloroacetic acid (DCA) Carcinogenic, corrosive, toxic

Dichloromethane (methylene chloride) Carcinogenic, mutagenic, teratogenic,toxic

Diethylamine (DEA) Corrosive, flammable liquid, toxic

Diethylpyrocarbonate (DEPC) Carcinogenic, toxic

Diethyl sulfate Carcinogenic, teratogenic, toxic See Basic Protocol 5

Diisopropyl fluorophosphate (DFP) Highly toxic, cholinesterase inhibitor,neurotoxin

See Basic Protocol 11

Dimethyldichlorosilane Corrosive, flammable liquid, toxic See Basic Protocol 3

Dimethyl sulfate (DMS) Carcinogenic, toxic See Basic Protocol 5

Dimethyl sulfoxide (DMSO) Flammable liquid, toxic Enhances absorptionthrough skin

Diphenylamine (DPA) Teratogenic, toxic

2,5-Diphenyloxazole (PPO) Toxic

Dithiothreitol (DTT) Toxic

Eosin B See Basic Protocol 2

Erythrosin B Carcinogenic, mutagenic See Basic Protocol 2

Ether Flammable liquid, toxic May form explosiveperoxides on standing; donot dry with NaOH or KOH

Ethidium bromide (EB) Mutagenic, toxic See Basic Protocol 2 or 6

Ethyl methanesulfonate (EMS) Carcinogenic, toxic See Basic Protocol 5

Fluorescein and derivatives Carcinogenic, toxic

5-Fluoro-2′-deoxyuridine (FUdR) Teratogenic, toxic

Fluoroorotic acid (FOA) Toxic

Formaldehyde Carcinogenic, flammable liquid,teratogenic, toxic

Formamide Teratogenic, toxic

Formic acid Corrosive, toxic May explode when heated>180°C in a sealed tube

Glutaraldehyde Corrosive, teratogenic, toxic

Guanidinium thiocyanate Toxic

Hoechst 33258 dye Mutagenic, toxic

Hydrochloric acid, concentrated Corrosive, teratogenic, toxic

Table A.1H.1 Commonly Used Hazardous Chemicalsa, continued


continued


A.1H.3


Hydrogen peroxide (30%) Carcinogenic, corrosive, mutagenic,oxidizer

Avoid bringing into contactwith organic materials,which may form explosiveperoxides; may decomposeviolently in contact withmetals, salts, or oxidizablematerials; see BasicProtocol 7

Hydroxylamine Corrosive, flammable, mutagenic,toxic

Explodes in air at >70°C

3-β-Indoleacrylic acid (IAA) Carcinogenic

Iodine Corrosive, toxic See Basic Protocol 8

Iodoacetamide Carcinogenic, mutagenic, toxic

Janus green B Carcinogenic, mutagenic See Basic Protocol 2

Lead compounds Carcinogenic, toxic

2-Mercaptoethanol (2-ME) Stench, toxic

Mercury compounds Teratogenic, toxic See Basic Protocol 9

Methionine sulfoximine (MSX) Teratogenic, toxic

Methotrexate (amethopterin) Carcinogenic, mutagenic, teratogenic,toxic

Methylene blue Mutagenic, toxic See Basic Protocol 2

Methyl methanesulfonate (MMS) Carcinogenic, toxic See Basic Protocol 5

Mycophenolic acid (MPA) Teratogenic, toxic

Neutral red Mutagenic See Basic Protocol 2

Nigrosin, water soluble See Basic Protocol 2

Nitric acid, concentrated Corrosive, oxidizer, teratogenic, toxic

Nitroblue tetrazolium (NBT) Toxic

Orcein, synthetic See Basic Protocol 2

Oxonols Toxic

Paraformaldehyde Toxic

Phenol Carcinogenic, corrosive, teratogenic,toxic

Readily absorbed throughthe skin

Phenylmethylsulfonyl fluoride (PMSF) Enzyme inhibitor See Basic Protocol 11

Phorbol 12-myristate 13-acetate (PMA) Carcinogenic, toxic

Phycoerythrins (PE) Toxic

Piperidine Flammable liquid, teratogenic, toxic

Potassium hydroxide, concentrated Corrosive, toxic Produces a highlyexothermic reaction whensolid is added to water

Propane sultone Carcinogenic, toxic See Basic Protocol 5

Propidium iodide (PI) Mutagenic See Basic Protocol 2 or 6

Pyridine Flammable liquid, toxic

Rhodamine and derivatives Toxic

Rose Bengal Carcinogenic, teratogenic See Basic Protocol 2

Safranine O Mutagenic See Basic Protocol 2



continued


A.1H.4

A.1H.3). No gloves resist all chemicals, and no gloves resist any chemicals indefinitely.Disposable gloves labeled “exam” or “examination” are primarily for protection frombiological materials (e.g., viruses, bacteria, feces, blood). They are not designed for andusually have not been tested for resistance to chemicals. Disposable gloves generally offerextremely marginal protection from chemical hazards in most cases and should beremoved immediately upon contamination before the chemical can pass through. Ifpossible, design handling procedures to eliminate or reduce potential for contamination.Never assume that disposable gloves will offer the same protection or even have the same

Sodium azide Carcinogenic, toxic Adding acid liberatesexplosive volatile, toxichydrazoic acid; can formexplosive heavy metalazides, e.g., with plumbingfixtures—do not dischargedown drain; see BasicProtocol 10

Sodium deoxycholate (Na-DOC) Carcinogenic, teratogenic, toxic

Sodium dodecyl sulfate (sodium lauryl sulfate,SDS)

Sensitizing, toxic

Sodium hydroxide, concentrated Corrosive, toxic A highly exothermicreaction ensues when thesolid is added to water

Sodium nitrite Carcinogenic

Sulfuric acid, concentrated Corrosive, oxidizer, teratogenic, toxic Reaction with water is veryexothermic; always addconcentrated sulfuric acid towater, never water to acid

SYTO dyes Toxic

Tetramethylammonium chloride (TMAC) Toxic

N,N,N′,N′-Tetramethyl-ethylenediamine (TEMED)

Corrosive, flammable liquid, toxic

Texas Red (sulforhodamine 101, acid chloride) Toxic

Toluene Flammable liquid, teratogenic, toxic

Toluidine blue O Mutagenic, toxic See Basic Protocol 2

Nα-p-Tosyl-L-lysine chloromethyl ketone (TLCK)

Toxic, enzyme inhibitor See Basic Protocol 11

N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK)

Toxic, mutagenic, enzyme inhibitor See Basic Protocol 11

Trichloroacetic acid (TCA) Carcinogenic, corrosive, teratogenic,toxic

Triethanolamine acetate (TEA) Carcinogenic, toxic

Trifluoroacetic acid (TFA) Corrosive, toxic

Trimethyl phosphate (TMP) Carcinogenic, mutagenic, teratogenic May explode on distillation

Trypan blue Carcinogenic, mutagenic, teratogenic See Basic Protocol 2

Xylenes Flammable liquid, teratogenic, toxicaFor extensive information on the hazards of these and other chemicals as well as cautionary details, see Bretherick (1986), O’Neil (2001), Furr (2000),Lewis (1999), Lunn and Sansone (1994a), and Bretherick et al. (1999).bCAUTION: These chemicals should be handled only in a chemical fume hood by knowledgeable workers equipped with eye protection, lab coat, andgloves. The laboratory should be equipped with a safety shower and eye wash. Additional protective equipment may be required.




A.1H.5


Table A.1H.2 Examples of Chemical Incompatibility

Chemical Incompatible with

Acetic acid Aldehydes, bases, carbonates, chromic acid, ethyleneglycol, hydroxides, hydroxyl compounds, metals, nitricacid, oxidizers, perchloric acid, peroxides, phosphates,permanganates, xylene

Acetone Acids, amines, concentrated nitric and sulfuric acidmixtures, oxidizers, plastics

Acetylene Copper, halogens, mercury, oxidizers, potassium, silverAlkali metals, alkaline earth metals

Acids, aldehydes, carbon dioxide, carbon tetrachloride orother chlorinated hydrocarbons, halogens, ketones,plastics, sulfur, water

Ammonia (anhydrous) Acids, aldehydes, amides, calcium hypochlorite,hydrofluoric acid, halogens, heavy metals, mercury,oxidizers, plastics, sulfur

Ammonium nitrate Acids, alkalis, chlorates, chloride salts, flammable andcombustible materials, metals, organic materials,phosphorus, reducing agents, sulfur, urea

Aniline Acids, aluminum, dibenzoyl peroxide, oxidizers, plasticsArsenical materials Any reducing agentAzides Acids, heavy metals, oxidizersBromine Acetaldehyde, alcohols, alkalis, amines, ammonia,

combustible materials, ethylene, fluorine, hydrogen,ketones (e.g., acetone, carbonyls), metals, petroleumgases, sodium carbide, sulfur

Calcium oxide Acids, ethanol, fluorine, organic materials, waterCarbon (activated) Alkali metals, calcium hypochlorite, halogens, oxidizersCarbon tetrachloride SodiumChlorates Acids, ammonium salts, finely divided organic or

combustible materials, powdered metals, sulfurChlorine Acetylene or other hydrocarbons, alcohols, ammonia,

benzene, butadiene, butane, combustible materials,ethylene, flammable compounds (e.g., hydrazine),hydrogen, hydrogen peroxide, iodine, metals, methane,nitrogen, oxygen, propane (or other petroleum gases),sodium carbide, sodium hydroxide

Chlorine dioxide Ammonia, hydrogen, hydrogen sulfide, mercury, methane,organic materials, phosphine, phosphorus, potassiumhydroxide, sulfur

Chromic acid, chromic oxide

Acetic acid, acetone, alcohols, alkalis, ammonia, bases,benzene, camphor, flammable liquids, glycerin (glycerol),hydrocarbons, metals, naphthalene, organic materials,phosphorus, plastics

Copper Acetylene, calcium, hydrocarbons, hydrogen peroxide,oxidizers

Cumene hydroperoxide Acids (organic or inorganic)Cyanides Acids, alkaloids, aluminum, iodine, oxidizers, strong basesFlammable liquids Ammonium nitrate, chromic acid, halogens, hydrogen

peroxide, nitric acid, oxidizing agents in general, oxygen,sodium peroxide

Fluorine All other chemicals

continued


A.1H.6

Safe Use ofHazardousChemicals

Hydrocarbons (liquid or gas)

See flammable liquids

Hydrocyanic acid Alkali, nitric acidHydrofluoric acid Ammonia, metals, organic materials, plastics, silica (glass,

including fiberglass), sodiumHydrogen peroxide All organics, most metals or their salts, nitric acid,

phosphorus, sodium, sulfuric acidHydrogen sulfide Acetylaldehyde, fuming nitric acid, metals, oxidizers,

sodium, strong basesHydroperoxide Reducing agentsHypochlorites Acids, activated carbonIodine Acetylaldehyde, acetylene, ammonia, hydrogen, metals,

sodiumMercury Acetylene, aluminum, amines, ammonia, calcium,

fulminic acid, lithium, oxidizers, sodiumNitric acid Acids, nitrites, metals, most organics, plastics, sodium,

sulfur, sulfuric acidNitrites AcidsNitroparaffins Amines, inorganic basesOxalic acid Mercury, oxidizers, silver, sodium chloriteOxygen All flammable and combustible materials, ammonia,

carbon monoxide, grease, metals, oil, phosphorus,polymers

Perchloric acid All organics, bismuth and alloys, dehydrating agents,grease, hydrogen halides, iodides, paper, wood

Peroxides, organic Acids (organic or mineral), avoid friction, store coldPhosphorus (white) Air, alkalis, oxygen, reducing agentsPotassium chlorate Acids, ammonia, combustible materials, fluorine,

hydrocarbons, metals, organic materials, reducing agents,sugars

Potassium perchlorate Alcohols, combustible materials, fluorine, hydrazine,metals, organic matter, reducing agents, sulfuric acid

Potassium permanganate Benzaldehyde, ethylene glycol, glycerin, sulfuric acidSelenides and tellurides Reducing agentsSilver Acetylene, ammonium compounds, fulminic acid, oxalic

acid, ozonides, peroxyformic acid, tartaric acidSodium Acids, carbon dioxide, carbon tetrachloride, hydrazine,

metals, oxidizers, waterSodium nitrate Acetic anhydride, acids, metals, organic matter,

peroxyformic acid, reducing agentsSodium peroxide Acetic anhydride, benzaldehyde, benzene, carbon

disulfide, ethyl acetate, ethyl or methyl alcohol, ethyleneglycol, furfural, glacial acetic acid, glycerin, hydrogensulfide, metals, methyl acetate, oxidizers, peroxyformicacid, phosphorus, reducing agents, sugars, water

Sulfides AcidsSulfuric acid Alcohols, bases, chlorates, perchlorates, permanganates of

potassium, lithium, sodium, magnesium, calcium

Table A.1H.2 Examples of Chemical Incompatibility, continued

Chemical Incompatible with


A.1H.7


Table A.1H.3 Chemical Resistance of Commonly Used Glovesa,b

Chemical Neoprene gloves Latex gloves Butyl gloves Nitrile gloves

*Acetaldehyde VG G VG GAcetic acid VG VG VG VG*Acetone G VG VG PAmmonium hydroxide VG VG VG VG*Amyl acetate F P F PAniline G F F P*Benzaldehyde F F G G*Benzene P P P FButyl acetate G F F PButyl alcohol VG VG VG VGCarbon disulfide F F F F*Carbon tetrachloride F P P G*Chlorobenzene F P F P*Chloroform G P P EChloronaphthalene F P F FChromic acid (50%) F P F FCyclohexanol G F G VG*Dibutyl phthalate G P G GDiisobutyl ketone P F G PDimethylformamide F F G GDioctyl phthalate G P F VGEpoxy resins, dry VG VG VG VG*Ethyl acetate G F G FEthyl alcohol VG VG VG VG*Ethyl ether VG G VG G*Ethylene dichloride F P F PEthylene glycol VG VG VG VGFormaldehyde VG VG VG VGFormic acid VG VG VG VGFreon 11, 12, 21, 22 G P F G*Furfural G G G GGlycerin VG VG VG VGHexane F P P GHydrochloric acid VG G G GHydrofluoric acid (48%) VG G G GHydrogen peroxide (30%) G G G GKetones G VG VG PLactic acid (85%) VG VG VG VGLinseed oil VG P F VGMethyl alcohol VG VG VG VGMethylamine F F G GMethyl bromide G F G F*Methyl ethyl ketone G G VG P*Methyl isobutylketone F F VG PMethyl methacrylate G G VG FMonoethanolamine VG G VG VG

continued


A.1H.8


properties as nondisposables. Select gloves carefully and always look for some evidencethat they will protect against the materials being used. Inspect all gloves before every usefor possible holes, tears, or weak areas. Never reuse disposable gloves. Clean reusablegloves after each use and dry carefully inside and out. Observe all common-senseprecautions—e.g., do not pipet by mouth, keep unauthorized persons away from hazard-ous chemicals, do not eat or drink in the lab, wear proper clothing in the lab (sandals,open-toed shoes, and shorts are not appropriate).

Order hazardous chemicals only in quantities that are likely to be used in a reasonabletime. Buying large quantities at a lower unit cost is no bargain if someone (perhapsyou) has to pay to dispose of surplus quantities. Substitute alcohol-filled thermometersfor mercury-filled thermometers, which are a hazardous chemical spill waiting tohappen.

Morpholine VG VG VG GNaphthalene G F F GNaphthas, aliphatic VG F F VGNaphthas, aromatic G P P G*Nitric acid G F F FNitric acid, red and whitefuming

P P P P

Nitropropane (95.5%) F P F FOleic acid VG F G VGOxalic acid VG VG VG VGPalmitic acid VG VG VG VGPerchloric acid (60%) VG F G GPerchloroethylene F P P GPhenol VG F G FPhosphoric acid VG G VG VGPotassium hydroxide VG VG VG VGPropyl acetate G F G Fi-Propyl alcohol VG VG VG VGn-Propyl alcohol VG VG VG VGSodium hydroxide VG VG VG VGStyrene (100%) P P P FSulfuric acid G G G GTetrahydrofuran P F F F*Toluene F P P FToluene diisocyanate F G G F*Trichloroethylene F F P GTriethanolamine VG G G VGTung oil VG P F VGTurpentine G F F VG*Xylene P P P FaPerformance varies with glove thickness and duration of contact. An asterisk indicates limited use. Abbreviations: VG,very good; G, good; F, fair; P, poor (do not use).

Table A.1H.3 Chemical Resistance of Commonly Used Glovesa,b, continued

Chemical Neoprene gloves Latex gloves Butyl gloves Nitrile gloves


A.1H.9


Although any number of chemicals commonly used in laboratories are toxic if usedimproperly, the toxic properties of a number of reagents require special mention. Chemicalsthat exhibit carcinogenic, corrosive, flammable, lachrymatory, mutagenic, oxidizing, tera-togenic, toxic, or other hazardous properties are listed in Table A.1H.1. Chemicals listedas carcinogenic range from those accepted by expert review groups as causing cancer inhumans to those for which only minimal evidence of carcinogenicity exists. No effort hasbeen made to differentiate the carcinogenic potential of the compounds in Table A.1H.1.Oxidizers may react violently with oxidizable material (e.g., hydrocarbons, wood, andcellulose). Before using any of these chemicals, thoroughly investigate all its charac-teristics. Material Safety Data Sheets are readily available; they list some hazards but varywidely in quality. A number of texts describing hazardous properties are listed at the endof this unit (see Literature Cited). In particular, Sax’s Dangerous Properties of IndustrialMaterials, 10th ed. (Lewis, 1999) and the Handbook of Reactive Chemical Hazards, 6thed. (Bretherick et al., 1999) give comprehensive listings of known hazardous properties;however, these texts list only the known properties. Many chemicals, especially fluoro-chromes, have been tested only partially or not at all. Prudence dictates that, unless thereis good reason for believing otherwise, all chemicals should be regarded as volatile, highlytoxic, flammable human carcinogens and should be handled with great care.

Waste should be segregated according to institutional requirements, for example, intosolid, aqueous, nonchlorinated organic, and chlorinated organic material, and shouldalways be disposed of in accordance with all applicable federal, state, and local regula-tions. Extensive information and cautionary details along with techniques for the disposalof chemicals in laboratories have been published (Bretherick, 1986; Lunn and Sansone,1994a; O’Neil, 2001; Furr, 2000). Some commonly used disposal procedures are outlinedin Basic Protocols 1 to 11. Incorporation of these procedures into laboratory protocolscan help to minimize waste disposal problems. Alternate Protocols 1 to 7 describedecontamination methods for some of the chemicals. Support Protocols 1 to 9 describeanalytical techniques that are used to verify that reagents have been decontaminated; withmodification, these assays may also be used to determine the concentration of a particularchemical.

DISPOSAL METHODS

A number of procedures for the disposal of hazardous chemicals are available; protocolsfor the disposal and decontamination of some hazardous chemicals commonly encoun-tered in molecular biology laboratories are listed in Table A.1H.4. These procedures arenecessarily brief; for full details consult the original references or a collection of theseprocedures (see Lunn and Sansone, 1994a).

CAUTION: These disposal methods should be carried out only in a chemical fume hoodby workers equipped with eye protection, a lab coat, and gloves. Additional protectiveequipment may be necessary.

BASICPROTOCOL 1

DISPOSAL OF BENZIDINE AND DIAMINOBENZIDINEBenzidine and diaminobenzidine can be degraded by oxidation with potassium perman-ganate (Castegnaro et al., 1985; Lunn and Sansone, 1991a). This protocol presents amethod for decontamination of benzidine and diaminobenzidine in bulk. This method canalso be adapted to the decontamination of benzidine and diaminobenzidine spills (seeAlternate Protocol 1). These compounds can also be removed from solution usinghorseradish peroxidase in the presence of hydrogen peroxide (see Alternate Protocol 2).Destruction and decontamination are >99%. Support Protocol 1 is used to test for thepresence of benzidine and diaminobenzidine.


A.1H.10


Materials

Benzidine or diaminobenzidine tetrahydrochloride dihydrate0.1 M HCl (for benzidine)0.2 M potassium permanganate: prepare immediately before use2 M sulfuric acidSodium metabisulfite10 M potassium hydroxide (KOH)

Additional reagents and equipment for testing for the presence of aromatic amines(see Support Protocol 1)

1. For each 9 mg benzidine, add 10 ml of 0.1 M HCl or for each 9 mg diaminobenzidinetetrahydrochloride dihydrate, add 10 ml water. Stir the solution until the aromaticamine has completely dissolved.

Table A.1H.4 Protocols for Disposal of Some Hazardous Chemicals

Protocol Disposal method for

Basic Protocol 1 Benzidine and diaminobenzidineAlternate Protocol 1 Spills of benzidine and diaminobenzidineAlternate Protocol 2 Aqueous solutions of benzidine and diaminobenzidineSupport Protocol 1 Analysis for benzidine and diaminobenzidine

Basic Protocol 2 Biological stainsAlternate Protocol 3 Large volumes of dilute biological stainsSupport Protocol 2 Analysis for biological stains

Basic Protocol 3 Silanes

Basic Protocol 4 Cyanide and cyanogen bromideSupport Protocol 3 Analysis for cyanide

Basic Protocol 5 Dimethyl sulfate, diethyl sulfate, methyl methanesulfonate, ethylmethanesulfonate, diepoxybutane, 1,3-propane sultone

Support Protocol 4 Analysis for dimethyl sulfate, diethyl sulfate, methyl methanesul-fonate, ethyl methanesulfonate, diepoxybutane, 1,3-propane sultone

Basic Protocol 6 Ethidium bromide and propidium iodideAlternate Protocol 4 Equipment contaminated with ethidium bromideAlternate Protocol 5 Ethidium bromide in isopropanol containing cesium chlorideAlternate Protocol 6 Ethidium bromide in alcoholsSupport Protocol 5 Analysis for ethidium bromide and propidium iodide

Basic Protocol 7 Hydrogen peroxide

Basic Protocol 8 Iodine

Basic Protocol 9 Mercury compoundsAlternate Protocol 7 Waste water containing mercurySupport Protocol 6 Analysis for mercury

Basic Protocol 10 Sodium azideSupport Protocol 7 Analysis for sodium azideSupport Protocol 8 Analysis for nitrite

Basic Protocol 11 Enzyme inhibitorsSupport Protocol 9 Analysis for enzyme inhibitors


A.1H.11


2. For each 10 ml of solution, add 5 ml freshly prepared 0.2 M potassium permanganateand 5 ml of 2 M sulfuric acid. Allow the mixture to stand for ≥10 hr.

3. Add sodium metabisulfite until the solution is decolorized.

4. Add 10 M KOH to make the solution strongly basic, pH >12.

CAUTION: This reaction is exothermic.

5. Dilute with 5 vol water and pass through filter paper to remove manganese com-pounds.

6. Test the filtrate for the presence of aromatic amines (i.e., benzidine or diaminobenzid-ine; see Support Protocol 1).

7. Neutralize the filtrate with acid and discard.

ALTERNATEPROTOCOL 1

DECONTAMINATION OF SPILLS INVOLVING BENZIDINE ANDDIAMINOBENZIDINE

Additional Materials (also see Basic Protocol 1)

Glacial acetic acid1:1 (v/v) 0.2 M potassium permanganate/2 M sulfuric acid: prepare immediately

before use

Absorbent material (e.g., paper towels, Kimwipes)High-efficiency particulate air (HEPA) vacuum (Fisher)


CAUTION: This procedure may damage painted surfaces and Formica.

1. Remove as much of the spill as possible using absorbent material and high-efficiencyparticulate air (HEPA) vacuuming.

2. Wet the surface with glacial acetic acid until all the amines are dissolved, then addan excess of freshly prepared 1:1 (v/v) 0.2 M potassium permanganate/2 M sulfuricacid to the spill area. Allow the mixture to stand ≥10 hr.

3. Ventilate the area and decolorize with sodium metabisulfite.

4. Mop up the liquid with paper towels. Squeeze the solution out of the towels and collectin a suitable container. Discard towels as hazardous solid waste.

5. Add 10 M KOH to make the solution strongly basic, pH ≥12.


6. Dilute with 5 vol water and filter through filter paper to remove manganese com-pounds.

7. Test the filtrate for the presence of aromatic amines (i.e., benzidine or diaminobenzid-ine; see Support Protocol 1).

8. Neutralize the filtrate with acid and discard it.

9. Verify complete decontamination by wiping the surface with a paper towel moistenedwith water and squeezing the liquid out of the towel. Test the liquid for the presenceof benzidine or diaminobenzidine (see Support Protocol 1). Repeat steps 1 to 9 asnecessary.


A.1H.12


ALTERNATEPROTOCOL 2

DECONTAMINATION OF AQUEOUS SOLUTIONS OF BENZIDINE ANDDIAMINOBENZIDINEThe enzyme horseradish peroxidase catalyzes the oxidation of the amine to a radicalwhich diffuses into solution and polymerizes. The polymers are insoluble and fall out ofsolution.


Aqueous solution of benzidine or diaminobenzidine1 N HCl or NaOH3% (v/v) hydrogen peroxideHorseradish peroxidase (see recipe)1:1 (v/v) 0.2 M potassium permanganate/2 M sulfuric acid5% (w/v) ascorbic acidPorous glass filter or Sorvall GLC-1 centrifuge or equivalent


1. Adjust the pH of the aqueous benzidine or diaminobenzidine solution to 5 to 7 with1 N HCl or NaOH as required and dilute so the concentration of aromatic amines is≤100 mg/liter.

2. For each liter of solution, add 3 ml of 3% hydrogen peroxide and 300 U horseradishperoxidase. Let the mixture stand 3 hr.

3. Remove the precipitate by filtering the solution through a porous glass filter or bycentrifuging 5 min at room temperature in a benchtop centrifuge to pellet theprecipitate.

The precipitate is mutagenic and should be treated as hazardous waste.

4. Immerse the porous glass filter in 1:1 (v/v) 0.2 M potassium permanganate/2 Msulfuric acid. Clean the filter in a conventional fashion and discard potassiumpermanganate/sulfuric acid solution as described for benzidine and diaminobenzid-ine (see Basic Protocol 1).

5. For each liter of filtrate, add 100 ml of 5% ascorbic acid.

6. Test the filtrate for the presence of aromatic amines (see Support Protocol 1).

7. Discard the decontaminated filtrate.

SUPPORTPROTOCOL 1

ANALYTICAL PROCEDURES TO DETECT BENZIDINE ANDDIAMINOBENZIDINEReversed-phase HPLC (Snyder et al., 1997) is used to test for the presence of aromaticamines. The limit of detection is 1 µg/ml for benzidine and 0.25 µg/ml for diaminobenzid-ine.

Materials

Decontaminated aromatic amine solution10:30:20 (v/v/v) acetonitrile/methanol/1.5 mM potassium phosphate buffer (1.5

mM K2HPO4/1.5 mM KH2PO4) (benzidine) or 75:25 (v/v) methanol/1.5 mMpotassium phosphate buffer (diaminobenzidine)

250-mm × 4.6-mm-i.d. Microsorb C-8 reversed-phase HPLC column (Varian) orequivalent

Additional reagents and equipment for reversed-phase liquid chromatography(Snyder et al., 1997)


A.1H.13


Analyze the decontaminated aromatic amine solution by reversed-phase HPLC using a250-mm × 4.6-mm-i.d. Microsorb C-8 column or equivalent. To detect benzidine, elutewith 10:30:20 (v/v/v) acetonitrile/methanol/1.5 mM potassium phosphate buffer at a flowrate of 1.5 ml/min and UV detection at 285 nm. To detect diaminobenzidine, elute with75:25 (v/v) methanol/1.5 mM potassium phosphate buffer at a flow rate of 1 ml/min andUV detection at 300 nm.

BASICPROTOCOL 2

DISPOSAL OF BIOLOGICAL STAINS

Biological stains (Table A.1H.5), as well as ethidium bromide and propidium iodide, canbe removed from solution using the polymeric resin Amberlite XAD-16. The decontami-nated solution may be disposed of as nonhazardous aqueous waste and the resin ashazardous solid waste. The volume of contaminated resin generated is much smaller thanthe original volume of the solution of biological stain, so the waste disposal problem isgreatly reduced. The final concentration of any remaining stain should be less than thelimit of detection (see Support Protocol 2 and Table A.1H.5). In each case decontamina-tion should be >99%. This protocol describes a method for batch decontamination inwhich the resin is stirred in the solution to be decontaminated and removed by filtrationat the end of the reaction time. Large volumes of biological stain can be decontaminatedusing a column (see Alternate Protocol 3). For full details refer to the original literature(Lunn and Sansone, 1991b) or a compilation (Lunn and Sansone, 1994a).

Table A.1H.5 Decontamination of Biological Stains

CompoundTime required for

completedecontamination

Volume of solution(ml) decontaminated

per gram resin

Acridine orange 18 hr 20Alcian blue 8GX 10 min 500Alizarin red S 18 hr 5Azure A 10 min 80Azure B 10 min 80Brilliant blue R 2 hr 80Congo red 2 hr 40Coomassie brilliant blue G 2 hr 80Cresyl violet acetate 2 hr 40Crystal violet 30 min 200Eosin B 30 min 40Erythrosin B 18 hr 10Ethidium bromide 4 hr 20Janus green B 30 min 80Methylene blue 30 min 80Neutral red 10 min 500Nigrosin 2 hr 80Orcein 2 hr 200Propidium iodide 2 hr 20Rose Bengal 3 hr 20Safranine O 1 hr 20Toluidine blue O 30 min 80Trypan blue 2 hr 40


A.1H.14


Materials

Amberlite XAD-16 resin (Supelco)100 µg/ml biological stain in water

Additional reagents and equipment for testing for the presence of biological stain(see Support Protocol 2)

For batch decontamination of 20 ml stain1a. Add 1 g Amberlite XAD-16 to 20 ml of 100 µg/ml biological stain in water.

For aqueous solutions having stain concentrations other than 100 g/ml, use proportion-ately greater or lesser amounts of resin to achieve complete decontamination.

For solutions of erythrosin B, use 2 g Amberlite XAD-16 for 20 ml stain.

2a. Stir the mixture for at least the time indicated in Table A.1H.5.

For batch decontamination of larger volumes of stain1b. Add 1 g Amberlite XAD-16 to the volume of a 100 µg/ml biological stain in water

indicated in Table A.1H.5.

2b. Stir the mixture for at least 18 hr.

3. Remove the resin by filtration through filter paper.

4. Test the filtrate for the presence of the biological stain (see Support Protocol 2).

5. Discard the resin as hazardous solid waste and the decontaminated filtrate as liquidwaste.

ALTERNATEPROTOCOL 3

CONTINUOUS-FLOW DECONTAMINATION OF AQUEOUSSOLUTIONS OF BIOLOGICAL STAINS

For treating large volumes of dilute aqueous solutions of biological stains (Table A.1H.5),it is possible to put the resin in a column and run the contaminated solution through thecolumn in a continuous-flow system (Lunn et al., 1994). Limited grinding of the AmberliteXAD-16 resin increases its efficiency.


25 µg/ml biological stain in waterMethanol (optional)

300-mm × 11-mm-i.d. glass chromatography column fitted with threaded adaptersand flow-regulating valves at top and bottom nut and insert connectors, andinsertion tool (Ace Glass) or 300-mm × 15-mm-i.d. glass chromatographycolumn (Spectrum 124010, Fisher)

Glass wool1.5-mm-i.d. × 0.3-mm-wall Teflon tubingWaring blender (optional)Rubber stopper fitted over a pencilQG 20 lab pump (Fluid Metering)

Additional reagents and equipment for testing for the presence of biological stain(see Support Protocol 2)

Using a slurry of Amberlite XAD-161a. Prepare a 300-mm × 11-mm-i.d. glass chromatography column. To prevent clogging

of the column outlet, place a small plug of glass wool at the bottom of the chroma-tography column. Connect 1.5-mm-i.d. × 0.3-mm wall Teflon tubing to the adaptersusing nut and insert connectors. Attach the tubing using an insertion tool.


A.1H.15


2a. Mix 10 g Amberlite XAD-16 and 25 ml water in a beaker and stir 5 min to wet theresin.

Using a finely ground Amberlite XAD-16 slurry1b. Prepare a 300-mm × 15-mm-i.d. glass chromatography column. To prevent clogging

of the column outlet, place a small plug of glass wool at the bottom of the chroma-tography column.

2b. Grind 20 g Amberlite XAD-16 with 200 ml water for exactly 10 sec in a Waringblender.

3. Pour the resin slurry into the column through a funnel. As the resin settles, tap thecolumn with a rubber stopper fitted over a pencil to encourage even packing. Attacha QG 20 lab pump.

4. Pump the 25-µg/ml biological stain solution through the column at 2 ml/min.

Alternatively, gravity flow coupled with periodic adjustment of the flow-regulating valvecan be used.

5. Check the effluent from the column for the presence of biological stain (see SupportProtocol 2). Stop the pump when stain is detected.

Table A.1H.6 lists breakthrough volumes at different detection levels for a number ofbiological stains.

6. Discard the decontaminated effluent and the contaminated resin appropriately.

7. Many biological stains can be washed off the resin with methanol so the resin can bereused. Discard the methanol solution of stain as hazardous organic liquid waste.

Table A.1H.6 Breakthrough Volumes for Continuous-FlowDecontamination of Biological Stains

CompoundBreakthrough volume (ml)

Limit ofdetection 1 ppm 5 ppm

Acridine orange 465 >990 >990Alizarin red S 120 150 240Azure A 615 810 >975Azure B 630 882 >1209Cresyl violet acetate 706 >1396 >1396Crystal violet 1020 >1630 >1630Ethidium bromide 260 312 416Janus green B 170 650 >870Methylene blue 420 645 1050Neutral red >2480 >2480 >2480Safranine O 365 438 584Toluidine blue O 353 494 606


A.1H.16


SUPPORTPROTOCOL 2

ANALYTICAL PROCEDURES TO DETECT BIOLOGICAL STAIN

Depending on the biological stain, the filtrate or eluate from the decontaminationprocedure can be analyzed using either UV absorption (A) or fluorescence detection (F).

Materials

Filtrate or eluate from biological stain decontamination (see Basic Protocol 2 or Alternate Protocol 3)

pH 5 buffer (see recipe)1 M KOH solution20 µg/ml calf thymus DNA in TBE electrophoresis buffer, pH 8.1 (APPENDIX 2A)

Test the filtrate or eluate from the biological stain decontamination procedure using theappropriate method as indicated in Table A.1H.7.

Traces of acid or base on the resin may induce color changes in the stain. Accordingly,with cresyl violet acetate or neutral red, mix aliquots of the filtrate with 1 vol pH 5 bufferbefore analyzing. With alizarin red S and orcein, mix aliquots of the filtrate with 1 vol of 1M KOH solution before analyzing.

Increase the fluorescence of solutions of acridine orange, ethidium bromide, and propidiumiodide by mixing an aliquot of the filtrate with an equal volume of 20 g/ml calf thymusDNA in TBE electrophoresis buffer, pH 8.1. Let the solution stand 15 min before measuringthe fluorescence.

Table A.1H.7 Methods for Detecting Biological Stainsa

Compound Reagentb Procedure Wavelength(s)(nm) Limit ofdetection (ppm)

Acridine orange DNA solution F ex 492, em 528 0.0032Alcian blue 8GX A 615 0.9Alizarin red S 1 M KOH A 556 0.46Azure A A 633 0.15Azure B A 648 0.13Brilliant blue R A 585 1.0Congo red A 497 0.25Coomassie brilliant blue G

A 610 1.7

Cresyl violet acetate pH 5 buffer F ex 588, em 618 0.021Crystal violet A 588 0.1Eosin B A 514 0.21Erythrosin B F ex 488, em 556 0.025Ethidium bromide F ex 540, em 590 0.05Janus green B A 660 0.6Methylene blue A 661 0.13Neutral red pH 5 buffer A 540 0.6Nigrosin A 570 0.8Orcein 1 M KOH A 579 1.15Propidium iodide DNA solution F ex 350, em 600 0.1Rose Bengal F ex 520, em 576 0.04Safranine O F ex 460, em 582 0.03Toluidine blue O A 626 0.2Trypan blue A 607 0.22aAbbreviations: A, absorbance; em, emission; ex, excitation; F, fluorescencebSee Support Protocol 2


A.1H.17


BASICPROTOCOL 3

DISPOSAL OF CHLOROTRIMETHYLSILANE ANDDICHLORODIMETHYLSILANESilane-containing compounds are hydrolyzed to hydrochloric acid and polymeric silicon-containing material (Patnode and Wilcock, 1946).

1. Hydrolyze silane-containing compounds by cautiously adding 5 ml silane to 100 mlvigorously stirred water in a flask. Allow the resulting suspension to settle.

2. Remove any insoluble material by filtration and discard it with the solid or liquidhazardous waste.

3. Neutralize the aqueous layer with base and discard it.

BASICPROTOCOL 4

DISPOSAL OF CYANIDES AND CYANOGEN BROMIDEInorganic cyanides (e.g., NaCN) and cyanogen bromide (CNBr) are oxidized by sodiumhypochlorite (NaOCl; e.g., Clorox) in basic solution to the much less toxic cyanate ion(Lunn and Sansone, 1985a). Destruction is >99.7%.

Materials

Cyanide (e.g., NaCN) or cyanogen bromide (CNBr)1 M NaOH5.25% (v/v) sodium hypochlorite (NaOCl; i.e., standard household bleach)

Additional reagents and equipment for testing for the presence of cyanide (seeSupport Protocol 3)

1. Dissolve cyanide in water to give a concentration ≤25 mg/ml or dissolve CNBr inwater to give a concentration ≤60 mg/ml.

If necessary, dilute aqueous solutions so the concentration of NaCN or CNBr does notexceed the limit.

2. Mix 1 vol NaCN or CNBr solution with 1 vol 1 M NaOH and 2 vol fresh 5.25%NaOCl. Stir the mixture 3 hr.

IMPORTANT NOTE: With age bleach may become ineffective; use of fresh bleach isstrongly recommended.

3. Test the reaction mixture for the presence of cyanide (see Support Protocol 3).

4. Neutralize the reaction mixture and discard it.

SUPPORTPROTOCOL 3

ANALYTICAL PROCEDURE TO DETECT CYANIDEThis protocol is used to detect cyanide or cyanogen bromide at ≥3 µg/ml.

Materials

Cyanide or cyanogen bromide decontamination reaction mixture (see BasicProtocol 4)

Phosphate buffer (see recipe)10 mg/ml sodium ascorbate in water: prepare fresh daily100 mg/liter NaCN in water: prepare fresh weekly10 mg/ml chloramine-T in water: prepare fresh dailyCyanide detection reagent (see recipe)Sorvall GLC-1 centrifuge or equivalent


A.1H.18


1. If necessary to remove suspended solids, centrifuge two 1-ml aliquots of the cyanideor cyanogen bromide decontamination reaction mixture 5 min in a benchtop centri-fuge, room temperature. Add each supernatant to 4 ml phosphate buffer in separatetubes.

2. If an orange or yellow color appears, add 10 mg/ml freshly prepared sodium ascorbatedropwise until the mixture is colorless, but do not add more than 2 ml.

3. Add 200 µl of 100 mg/liter NaCN to one reaction mixture (control solution).

4. Add 1 ml freshly prepared 10 mg/ml chloramine-T to each tube. Shake the tubes andlet them stand 1 to 2 min.

5. Add 1 ml cyanide detection reagent, shake, and let stand 5 min.

A blue color indicates the presence of cyanide. If destruction has been complete and theanalytical procedure has been carried out correctly, the treated reaction mixture should becolorless and the control solution, which contains NaCN, should be blue.

6. Centrifuge tubes 5 min, room temperature, if necessary to remove suspended solids.Measure the absorbance at 605 nm with appropriate standards and blanks.

BASICPROTOCOL 5

DISPOSAL OF DIMETHYL SULFATE, DIETHYL SULFATE, METHYLMETHANESULFONATE, ETHYL METHANESULFONATE,DIEPOXYBUTANE, AND 1,3-PROPANE SULTONE

Dimethyl sulfate is hydrolyzed by base to methanol and methyl hydrogen sulfate (Lunnand Sansone, 1985b). Subsequent hydrolysis of methyl hydrogen sulfate to methanol andsulfuric acid is slow. Methyl hydrogen sulfate is nonmutagenic and a very poor alkylatingagent. The other compounds can be hydrolyzed with base in a similar fashion (Lunn andSansone, 1990a). Destruction is >99%. A method to verify that decontamination iscomplete is also provided (see Support Protocol 4).

NOTE: The reaction times given in the protocol should give good results; however,reaction time may be affected by such factors as the size and shape of the flask and therate of stirring. The presence of two phases indicates that the reaction is not complete,and stirring should be continued until the reaction mixture is homogeneous.

Materials

Dimethyl sulfate, diethyl sulfate, methyl methanesulfonate, ethylmethanesulfonate, diepoxybutane, or 1,3-propane sultone

5 M NaOH

Additional reagents and equipment for testing for the presence of dimethyl sulfate,diethyl sulfate, methyl methanesulfonate, ethyl methanesulfonate,diepoxybutane, or 1,3-propane sultone (see Support Protocol 4)

For bulk quantities of dimethyl sulfate1a. Add 100 ml dimethyl sulfate to 1 liter of 5 M NaOH. Stir the reaction mixture.

2a. Fifteen minutes after all the dimethyl sulfate has gone into solution, neutralize thereaction mixture with acid.

For bulk quantities of diethyl sulfate1b. Add 100 ml diethyl sulfate to 1 liter of 5 M NaOH. Stir the reaction mixture 24 hr.

2b. Neutralize the reaction mixture with acid.


A.1H.19


For bulk quantities of methyl methanesulfonate, ethyl methanesulfonate,diepoxybutane, and 1,3-propane sultone1c. Add 1 ml methyl methanesulfonate, ethyl methanesulfonate, or diepoxybutane, or 1

g of 1,3-propane sultone to 10 ml of 5 M NaOH. Stir the reaction mixture 1 hr for1,3-propane sultone, 2 hr for methyl methanesulfonate, 22 hr for diepoxybutane, or24 hr for ethyl methanesulfonate.

2c. Neutralize the reaction mixture with acid.

3. Test the reaction mixture for the presence of the original compound (see SupportProtocol 4).

4. Discard the decontaminated reaction mix.

SUPPORTPROTOCOL 4

ANALYTICAL PROCEDURE TO DETECT THE PRESENCE OF DIMETHYLSULFATE, DIETHYL SULFATE, METHYL METHANESULFONATE, ETHYLMETHANESULFONATE, DIEPOXYBUTANE, AND 1,3-PROPANE SULTONEThis protocol is used to verify decontamination of solutions containing dimethyl sulfate,diethyl sulfate, methyl methanesulfonate, ethyl methanesulfonate, diepoxybutane, or1,3-propane sultone. The detection limit for this assay is 40 µg/ml for dimethyl sulfate,108 µg/ml for diethyl sulfate, 84 µg/ml for methyl methanesulfonate, 1.1 µg/ml for ethylmethanesulfonate, 360 µg/ml for diepoxybutane, and 264 µg/ml for 1,3-propane sultone.

Materials

Reaction mixture containing dimethyl sulfate, diethyl sulfate, methylmethanesulfonate, ethyl methanesulfonate, diepoxybutane, or 1,3-propanesultone

98:2 (v/v) 2-methoxyethanol/acetic acid5% (w/v) 4-(4-nitrobenzyl)pyridine in 2-methoxyethanolPiperidine2-Methoxyethanol

1. Dilute an aliquot of the reaction mixture with 4 vol water.

2. Add 100 µl diluted reaction mixture to 1 ml of 98:2 (v/v) 2-methoxyethanol/aceticacid. Swirl to mix.

3. Add 1 ml of 5% (w/v) 4-(4-nitrobenzyl)pyridine in 2-methoxyethanol. Heat 10 minat 100°C, then cool 5 min in ice.

4. Add 0.5 ml piperidine and 2 ml of 2-methoxyethanol.

5. Measure the absorbance of the violet reaction mixture at 560 nm against an appro-priate blank.

The absorbance of a decontaminated solution should be 0.000.

BASICPROTOCOL 6

DISPOSAL OF ETHIDIUM BROMIDE AND PROPIDIUM IODIDE

Ethidium bromide and propidium iodide in water and buffer solutions may be degradedby reaction with sodium nitrite and hypophosphorous acid in aqueous solution (Lunn andSansone, 1987); destruction is >99.87%. This reaction may also be used to decontaminateequipment contaminated with ethidium bromide (see Alternate Protocol 4; Lunn andSansone, 1989) and to degrade ethidium bromide in organic solvents (see AlternateProtocol 5 and Alternate Protocol 6; Lunn and Sansone, 1990b). Ethidium bromide andpropidium iodide may also be removed from solution by adsorption onto AmberliteXAD-16 resin (see Basic Protocol 2).


A.1H.20


Materials

Ethidium bromide– or propidium iodide–containing solution in water, buffer,or 1 g/ml cesium chloride

5% (v/v) hypophosphorous acid: prepare fresh daily by diluting commercial50% reagent 1/10

0.5 M sodium nitrite: prepare fresh dailySodium bicarbonate

Additional reagents and equipment for testing for the presence of ethidiumbromide or propidium iodide (see Support Protocol 5)

1. If necessary, dilute the ethidium bromide– or propidium iodide–containing solutionso the concentration of ethidium bromide or propidium iodide is ≤0.5 mg/ml.

2. For each 100 ml solution, add 20 ml of 5% hypophosphorous acid solution and 12ml of 0.5 M sodium nitrite. Stir briefly and let stand 20 hr.

3. Neutralize the reaction mixture by adding sodium bicarbonate until the evolution ofgas ceases.

4. Test the reaction mixture for the presence of ethidium bromide or propidium iodide(see Support Protocol 5).

5. Discard the decontaminated reaction mixture.

ALTERNATEPROTOCOL 4

DECONTAMINATION OF EQUIPMENT CONTAMINATED WITHETHIDIUM BROMIDE

Glass, stainless steel, Formica, floor tile, and the filters of transilluminators have beensuccessfully decontaminated using this protocol. No change in the optical properties ofthe transilluminator filter could be detected, even after a number of decontaminationcycles.

Materials

Equipment contaminated with ethidium bromideDecontamination solution (see recipe)Sodium bicarbonate

Additional reagents and equipment for testing for the presence of ethidiumbromide (see Support Protocol 5)

1. Wash the equipment contaminated with ethidium bromide once with a paper towelsoaked in decontamination solution.

The pH of the decontamination solution is 1.8. If this would be too corrosive for the surfaceto be decontaminated, wash with a paper towel soaked in water instead.

2. Wash the surface five times with paper towels soaked in water using a fresh toweleach time.

3. Soak all the towels 1 hr in decontamination solution.

4. Neutralize the decontamination solution by adding sodium bicarbonate until theevolution of gas ceases.

5. Test the decontamination solution for the presence of ethidium bromide (see SupportProtocol 5).

6. Discard the decontamination solution and the paper towels as nonhazardous liquidand solid wastes.


A.1H.21


ALTERNATEPROTOCOL 5

DECONTAMINATION OF ETHIDIUM BROMIDE IN ISOPROPANOLSATURATED WITH CESIUM CHLORIDE

Materials

Ethidium bromide in isopropanol saturated with cesium chlorideDecontamination solution (see recipe)Sodium bicarbonate

Additional reagents and equipment for testing for the presence of ethidiumbromide (see Support Protocol 5)

1. If necessary, dilute the ethidium bromide in isopropanol saturated with cesiumchloride so the concentration of ethidium bromide is ≤1 mg/ml.

2. For each volume of ethidium bromide solution, add 4 vol decontamination solution.Stir the reaction mixture 20 hr.

3. Neutralize the reaction mixture by adding sodium bicarbonate until the evolution ofgas ceases.

4. Test the reaction mixture for the presence of ethidium bromide (see Support Protocol5).

5. Discard the decontaminated solution.

ALTERNATEPROTOCOL 6

DECONTAMINATION OF ETHIDIUM BROMIDE IN ISOAMYLALCOHOL AND 1-BUTANOL

Materials

Ethidium bromide in isoamyl alcohol or 1-butanolDecontamination solution (see recipe)Activated charcoalSodium bicarbonateSeparatory funnel

Additional reagents and equipment for testing for the presence of ethidium bromide

1. If necessary, dilute the ethidium bromide in isoamyl alcohol or 1-butanol so theconcentration is ≤1 mg/ml final.

2. For each volume of ethidium bromide solution, add 4 vol decontamination solution.Stir the two-phase reaction mixture rapidly for 72 hr.

3. For each 100 ml of reaction mixture, add 2 g activated charcoal. Stir another 30 min.

4. Filter the reaction mixture.

5. Neutralize the filtrate by adding sodium bicarbonate until the evolution of gas ceases.Separate the layers using a separatory funnel.

More alcohol may tend to separate from the aqueous layer on standing.

6. Test the alcohol and aqueous layers for the presence of ethidium bromide.

7. Discard the alcohol and aqueous layers appropriately. Discard the activated charcoalas solid waste.

The aqueous layer contains 4.6% 1-butanol or 2.3% isoamyl alcohol.


A.1H.22


SUPPORTPROTOCOL 5

ANALYTICAL PROCEDURE TO DETECT ETHIDIUM BROMIDE ORPROPIDIUM IODIDEThis protocol is used to verify that solutions no longer contain ethidium bromide orpropidium iodide. The limits of detection are 0.05 parts per million (ppm) for ethidiumbromide and 0.1 ppm for propidium iodide.

Materials

Reaction mixture containing ethidium bromide or propidium iodideTBE buffer, pH 8.1 (APPENDIX 2A)20 µg/ml calf thymus DNA in TBE buffer, pH 8.1

1. Mix 100 µl reaction mixture containing ethidium bromide or propidium iodide with900 µl TBE buffer, pH 8.1.

2. Add 1 ml of 20 µg/ml calf thymus DNA in TBE, pH 8.1. Prepare a blank solution(100 µl water + 900 µl TBE + 1 ml of 20 µg/ml calf thymus DNA) and controlsolutions containing known quantities of ethidium bromide or propidium iodide. Letthe mixtures stand 15 min.

3. To detect ethidium bromide, measure the fluorescence with an excitation wavelengthof 540 nm and an emission wavelength of 590 nm. To detect propidium iodide,measure the fluorescence with an excitation wavelength of 350 nm and an emissionwavelength of 600 nm.

If a spectrophotofluorometer is not available, fluorescence of ethidium bromide can bequalitatively determined using a hand-held UV lamp on the long-wavelength setting (Lunnand Sansone, 1991c).

BASICPROTOCOL 7

DISPOSAL OF HYDROGEN PEROXIDEHydrogen peroxide can be reduced with sodium metabisulfite (Lunn and Sansone,1994b).

Materials

30% hydrogen peroxide10% (w/v) sodium metabisulfite10% (w/v) potassium iodide1 M HCl1% (w/v) starch indicator solution

1. Add 5 ml of 30% hydrogen peroxide to 100 ml of 10% sodium metabisulfite. Stir themixture at room temperature until the temperature starts to drop, indicating that thereaction is over.

2. Test for the presence of hydrogen peroxide by adding a few drops of the reactionmixture to an equal volume of 10% potassium iodide. Add a few drops of 1 M HClto acidify the reaction mixture, then add a drop of 1% starch indicator solution.

A deep blue color indicates the presence of excess oxidant. If necessary, add more 10%sodium metabisulfite until the starch test is negative.

3. Discard the decontaminated mixture.


A.1H.23


BASICPROTOCOL 8

REDUCTION OF IODINEIodine is reduced with sodium metabisulfite to iodide (Lunn and Sansone, 1994b).

Materials

Iodine10% (w/v) sodium metabisulfite1 M HCl1% (w/v) starch indicator solution

1. Add 5 g iodine to 100 ml of 10% sodium metabisulfite. Stir the mixture until theiodine has completely dissolved.

2. Acidify a few drops of the reaction mixture by adding a few drops of 1 M HCl. Add1 drop of 1% starch indicator solution.

A deep blue color indicates the presence of iodine. If reduction is not complete, add moresodium metabisulfite solution.

3. Dispose of the decontaminated solution.

BASICPROTOCOL 9

DISPOSAL OF MERCURY COMPOUNDSSolutions of mercuric acetate can be decontaminated using Dowex 50X8-100, a stronglyacidic gel-type ion-exchange resin with a sulfonic acid functionality. Solutions of mercu-ric chloride can be decontaminated using Amberlite IRA-400(Cl), a strongly basicgel-type ion-exchange resin with a quaternary ammonium functionality. The final con-centration of mercury is <3.8 ppm (Lunn and Sansone, 1994a). On a small scale it is mostconvenient to stir the resin in the solution to be decontaminated, but on a larger scale, orfor routine use, it may be more convenient to pass the solution through a column packedwith the resin. Although the volume of waste that must be disposed of is greatly reducedusing this technique, a small amount of waste (i.e., the resin contaminated with mercury)remains and must be discarded appropriately. Resin can be regenerated by washing withacid, but the concentrated metal-containing solution generated by this must also bedisposed of appropriately. Mercury may also be removed from laboratory waste waterusing a column of iron powder (see Alternate Protocol 7). Support Protocol 6 is used todetect the presence of mercury.

Materials

Solution containing ≤1600 ppm mercuric acetate or ≤1350 ppm mercuric chlorideDowex 50X8-100 ion-exchange resin or Amberlite IRA-400(Cl) ion-exchange

resin

Additional reagents and equipment to test for the presence of mercury (seeSupport Protocol 6)

1a. For mercuric acetate: For each 200 ml of solution containing ≤1600 ppm mercuricacetate, add 1 g Dowex 50X8-100 ion-exchange resin. Stir the mixture 1 hr, then filterthrough filter paper.

1b. For mercuric chloride: For each 200 ml of solution containing ≤1350 ppm mercuricchloride, add 1 g Amberlite IRA-400(Cl) ion-exchange resin. Stir the mixture 6 hr,then filter through filter paper.

The speed and efficiency of decontamination will depend on factors such as the size andshape of the flask and the rate of stirring.

3. Test the filtrate for the presence of mercury (see Support Protocol 6).

4. Discard the decontaminated filtrate and the mercury-containing resin appropriately.


A.1H.24


ALTERNATEPROTOCOL 7

DECONTAMINATION OF WASTE WATER CONTAINING MERCURY

Laboratory waste water that contains mercury is decontaminated by passing it through acolumn of iron powder. The mercury forms mercury amalgam and stays on the column.Some metallic mercury remains in solution but this can be removed by aeration. The finalconcentration of mercury is <5 ppb (Shirakashi et al., 1986).

Materials

Iron powder, 60 meshWaste water containing ≤2.5 ppm mercury6-mm-i.d. column

1. Pack a 6-mm-i.d. column with 1 g of 60-mesh iron powder.

Use a fresh column for each treatment.

2. Pass ≤2 liters of water containing ≤2.5 ppm of mercury through the column at a flowrate of 250 ml/hr.

Solutions containing a higher concentration of mercury may also be treated, but this willresult in a higher final concentration of mercury (e.g., treating a 100-ppm solution in thisfashion yielded 33 ppb final).

Some iron ends up in solution and can be removed by adjusting the pH to 8. The resultingprecipitated Fe(OH)3 can then be removed by filtration.

3. Aerate the resulting effluent to remove traces of metallic mercury and continueaeration 30 min after the last of the effluent has emerged from the column. Vent themetallic mercury removed from the solution by aeration into the fume hood or captureit in a mercury trap.

The effluent contains <5 ppb mercury. The presence of iodide or polypeptone may neces-sitate several treatments to reduce the mercury to an acceptable level.

SUPPORTPROTOCOL 6

ANALYTICAL PROCEDURE TO DETECT MERCURYAtomic absorption spectroscopy with detection at 253.7 nm, a slit width of 0.7 nm, anda limit of detection of 3.8 ppm can be used to determine the concentration of mercury insolution for experiments involving ion-exchange resins. A Hiranuma mercury metermodel HG-1 can be used for experiments involving iron powder.

BASICPROTOCOL 10

DISPOSAL OF SODIUM AZIDESodium azide can be oxidized by ceric ammonium nitrate (Manufacturing ChemistsAssociation, 1973) to nitrogen (Mason, 1967) or by nitrous acid (National ResearchCouncil, 1983) to nitrous oxide (Mason, 1967); destruction is >99.996%. Sodium azidein buffer solution may also be degraded by the addition of sodium nitrite (Lunn andSansone, 1994a). The reaction proceeds much more readily at low pH, but if sufficientsodium nitrite is added, it will proceed to completion even at high pH. At low pH, it maybe possible to completely degrade the azide present in the buffer with less than the amountof sodium nitrite indicated. However, the reaction mixture must be carefully checked toensure that no azide remains (see Support Protocol 7). At high pH it is possible forunreacted azide to remain in the presence of excess nitrite. Residual nitrite can be detectedusing Support Protocol 8.

CAUTION: Some toxic nitrogen dioxide may be produced as a by-product of thesereactions, so they should always be carried out in a chemical fume hood.


A.1H.25


Materials

Sodium azide or solution containing sodium azideCeric ammonium nitrate10% (w/v) potassium iodide1 M HCl1% (w/v) starch indicator solutionSodium nitrite4 M sulfuric acid

Additional reagents and equipment to test for the presence of sodium azide (seeSupport Protocol 7) or nitrite (see Support Protocol 8)

Decontamination using ceric ammonium nitrate1a. For each gram of sodium azide, add 9 g ceric ammonium nitrate to 30 ml of water,

and stir until it has dissolved.

2a. Dissolve each gram of sodium azide in 5 ml water. Add this solution to the cericammonium nitrate solution at the rate of 1 ml each min. Stir 1 hr.

If the reaction is carried out on a larger scale, an ice bath may be required for cooling.

3a. Check that the reaction is still oxidizing by adding a few drops of the reaction mixtureto an equal volume of 10% potassium iodide. Acidify the mixture with 1 drop 1 MHCl and add 1 drop 1% starch indicator solution.

The deep blue color of the starch-iodine complex indicates that excess oxidant is present.If excess oxidant is not present, add more ceric ammonium nitrate.

4a. Test for the presence of sodium azide (see Support Protocol 8).

5a. Discard the decontaminated reaction mixture.

Decontamination using sodium nitrite1b. For each 5 g sodium azide, dissolve 7.5 g sodium nitrite in 30 ml water.

2b. Dissolve each 5 g sodium azide in 100 ml water. Add the sodium nitrite solution withstirring. Slowly add 4 M sulfuric acid until the reaction mixture is acidic to litmus.Stir 1 hr.

CAUTION: It is important to add the sodium nitrite, then the sulfuric acid. Adding thesereagents in reverse order will generate explosive, volatile, toxic hydrazoic acid.

If the reaction is carried out on a large scale, an ice bath may be required for cooling.

3b. Check that there is excess nitrous acid in the reaction. Add a few drops of the reactionmixture to an equal volume of 10% potassium iodide. Acidify the mixture with 1 drop1 M HCl. Add 1 drop starch indicator solution.

The deep blue color of the starch-iodine complex indicates that excess nitrous acid ispresent. If excess nitrous acid is not present, add more sodium nitrite.

4b. If excess nitrous acid is present, test for the presence of sodium azide (see SupportProtocol 7).

5b. Discard the decontaminated reaction mixture.

Decontamination of sodium azide in buffer1c. If necessary, dilute the buffer solution with water so the concentration of sodium azide

is ≤1 mg/ml.

2c. For each 50 ml buffer solution add 5 g sodium nitrite. Stir the reaction 18 hr.


A.1H.26


3c. Test for the presence of sodium azide (see Support Protocol 7).

4c. Discard the decontaminated reaction solution.

SUPPORTPROTOCOL 7

ANALYTICAL PROCEDURES TO DETECT SODIUM AZIDE

Sodium azide is analyzed by reacting azide ion with 3,5-dinitrobenzoyl chloride to form3,5-dinitrobenzoyl azide, which can be detected by reversed-phase HPLC. The limit ofdetection of this assay is 0.2 µg/ml sodium azide. This protocol works only in the absenceof nitrite; verify that all of the nitrite has been destroyed by sulfamic acid by using themethod detailed later in this unit (see Support Protocol 8).

Materials

Reaction mixture from sodium azide treated with ceric ammonium nitrate orsodium nitrite

1 M KOHAcetonitrileSodium azide indicator solution (see recipe)0.2 M HCl20% (w/v) sulfamic acid3,5-dinitrobenzoyl chloride50:50 (v/v) acetonitrile/water

Sorvall GLC-1 centrifuge or equivalent25-cm × 4.6-mm-i.d. Microsorb C-8 reversed-phase HPLC column (Varian) or

equivalent


To analyze for azide in the presence of ceric salts1a. To a 10-ml aliquot of the reaction mixture from sodium azide treated with ceric

ammonium nitrate add 40 ml water. Add 5 ml of this diluted solution to 3 ml of 1 MKOH and mix by shaking.

If <3 ml of 1 M KOH is used, precipitation of ceric salts will not be complete.

2a. Centrifuge the mixture 5 min, room temperature.

3a. Remove 2 ml supernatant and add to 1 ml acetonitrile. Add 1 drop sodium azideindicator solution, add 0.2 M HCl dropwise until the mixture turns yellow, then add1 drop more.

To analyze for azide in the presence of nitrite1b. To 5 ml of the reaction mixture from sodium azide treated with sodium nitrite add ≥1

ml sulfamic acid to remove excess nitrite. Let stand ≥3 min.

More sulfamic acid solution may be required for strongly basic reaction mixtures or thosecontaining high concentrations of nitrite. Complete removal of nitrite can be checked byusing a modified Griess reagent (see Support Protocol 8).

At high pH the reaction between azide and nitrite is quite slow, so the presence of excessnitrite does not mean that all the azide has been degraded.

2b. Add 1 drop sodium azide indicator solution, then basify the mixture by adding 1 MKOH until it turns purple (typically, 3 to 10 ml are required).

3b. Add 2 ml acetonitrile. Add 0.2 M HCl dropwise until the mixture turns yellow, thenadd 1 drop more.

If >1 ml sulfamic acid is used, add 4 ml acetonitrile.


A.1H.27


4. Prepare a 10% (w/v) solution of 3,5-dinitrobenzoyl chloride in acetonitrile.

5. Add 50 µl of 10% dinitrobenzoyl chloride/acetonitrile to the reaction mix (step 3a or3b). Shake the mixture and let it stand ≥3 min.

Longer standing times have no effect on the HPLC analysis. However, it is crucial to usefreshly prepared 3,5-dinitrobenzoyl chloride solution within minutes of its preparation. Itis generally most convenient to prepare all the analytical samples with the fresh solutionat the beginning of the day and analyze them over the course of the day.

6. Analyze 20 µl of each reaction mixture by reversed-phase HPLC (Snyder et al., 1997)using a mobile phase of 50:50 (v/v) acetonitrile/water with a flow rate of 1 ml/minand UV detection at 254 nm.

The peak for 3,5-dinitrobenzoyl azide elutes at ∼9 min.

SUPPORTPROTOCOL 8

ANALYTICAL PROCEDURE TO DETECT NITRITE

This protocol uses a modified Griess reagent to test for the presence of nitrite. The limitof detection of this assay is 0.06 µg/ml nitrite. A similar procedure uses N-(1-naphthyl)-ethylenediamine (Cunniff, 1995).

Materials

α-Naphthylamine15% (v/v) aqueous acetic acidSulfanilic acid solution (see recipe)Reaction mixture treated to remove excess nitrite (see Support Protocol 7, step 1b)

Table A.1H.8 Conditions for the Destruction of Enzyme Inhibitors

Compound Concentration Solvent Solution: 1 M NaOH Time

AEBSF 1 mM Buffer(pH 5.0-8.0) 1:0.1 1 hrAEBSF 20 mM DMSO 1:10 24 hrAEBSF 20 mM Isopropanol 1:10 24 hrAPMSF 2.5 mM Buffer(pH 5.0-8.0) 1:0.1 1 hrAPMSF 25 mM DMSO 1:5 24 hrAPMSF 25 mM 50:50 isopropanol:pH 3 buffer 1:5 24 hrAPMSF 100 mM Water 1:5 24 hrDFP 10 mM Buffer (pH 6.4-7.2) 1:0.2 18 hrDFP 200 mM DMF 1:2 18 hrDFP pure — 1:25 1 hrDFP 10 mM Water 1:0.2 18 hrPMSF 10 mM Buffer (pH 5.0-8.0) 1:0.1 1 hrPMSF 100 mM DMSO 1:5 24 hrPMSF 100 mM Isopropanol 1:5 24 hrTLCK 1 mM Buffer (pH 5.0-8.0) 1:0.1 18 hrTLCK 5 mM DMSO 1:5 18 hrTLCK 5 mM Water 1:0.1 18 hrTPCK 1 mM Buffer (pH 5.0-8.0) 1:0.1 18 hrTPCK 1 mM DMSO 1:0.1 18 hrTPCK 1 mM Isopropanol 1:0.1 18 hr


A.1H.28


1. Prepare the modified Griess reagent by boiling 0.1 g α-naphthylamine in 20 ml wateruntil it dissolves. While the solution is still hot, pour it into 150 ml of 15% aqueousacetic acid. Add 150 ml sulfanilic acid solution.

This reagent should be stored at room temperature in a brown bottle.

CAUTION: α-Naphthylamine is a carcinogen.

2. Add 3 ml of the reaction mixture treated to remove excess nitrite to 1 ml modifiedGriess reagent. Let stand 6 min at room temperature.

3. Measure the absorbance at 520 nm against a suitable blank.

BASICPROTOCOL 11

DISPOSAL OF ENZYME INHIBITORS

The enzyme inhibitors p-amidinophenylmethanesulfonyl fluoride (APMSF), 4-(2-ami-noethyl)benzenesulfonyl fluoride (AEBSF), phenylmethylsulfonyl fluoride (PMSF;Lunn and Sansone, 1994c), diisopropyl fluorophosphate (DFP; Lunn and Sansone,1994d), Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK), and N-p-tosyl-L-phenylalan-ine chloromethyl ketone (TPCK; Lunn and Sansone, 1994c) may be degraded by reactionwith 1 M NaOH. Destruction is >99.8% (except TPCK >98.3%). The exact reactionconditions depend on the solvent (see Table A.1H.8). The solutions that were decontami-nated are representative of those described in the literature.

Materials

Solutions of APMSF, AEBSF, PMSF, DFP, TLCK, or TPCK in buffer, DMSO,isopropanol, or water

1 M NaOHGlacial acetic acid

Additional reagents and equipment for testing for the presence of the enzymeinhibitors (see Support Protocol 9)

1. If necessary, dilute the solutions with the same solvent so that the concentrationsgiven in Table A.1H.8 are not exceeded.

Bulk quantities of AEBSF, PMSF, and TPCK may be dissolved in isopropanol and bulkquantities of APMSF and TLCK may be dissolved in water at the concentrations shown inTable A.1H.8. Bulk quantities of DFP (a liquid) may be mixed directly with 1 M NaOH,taking care to make sure that all the DFP has mixed thoroughly, in the ratio shown in TableA.1H.8 (e.g., 40 l DFP with 1 ml of 1 M NaOH).

2. Add 1 M NaOH so that the ratio of solution to 1 M NaOH is that listed in TableA.1H.8.

3. Shake to ensure complete mixing, check that the solution is strongly basic (pH ≥12),and allow to stand for the time given in Table A.1H.8.

4. Neutralize the reaction mixture with acetic acid and test for the presence of residualenzyme inhibitor (see Support Protocol 9).

5. Discard the decontaminated reaction mixture.


A.1H.29


SUPPORTPROTOCOL 9

ANALYTICAL PROCEDURES TO DETECT ENZYME INHIBITORS

DFP can be determined using a complex procedure involving the inhibition of chymotryp-sin activity. For more information, refer to Lunn and Sansone (1994d). A gas chroma-tographic method has also been described by Degenhardt-Langelaan and Kientz (1996).AEBSF, APMSF, PMSF, TLCK, and TPCK may be determined by reversed-phase HPLC(Snyder et al., 1997). The chromatographic conditions and limits of detection are shownin Table A.1H.9 (Lunn and Sansone, 1994c).

Materials

Decontaminated enzyme inhibitor solutionsAcetonitrile (HPLC grade)Water (HPLC grade)0.1% (v/v) trifluoroacetic acid in water10 mM phosphate buffer, pH 7250-mm × 4.6 mm-i.d. Microsorb C-8 reversed-phase HPLC column (Varian) or

equivalent


Analyze the decontaminated enzyme inhibitor solutions by reversed-phase HPLC usinga 250-mm × 4.6-mm-i.d. Microsorb C-8 reversed-phase column, or equivalent, using theconditions shown in Table A.1H.9. In each case, the injection volume was 20 µl, theseparation occurred at ambient temperature, and the flow rate was 1 ml/min. Check theanalytical procedures by spiking an aliquot of the acidified reaction mixture with a smallquantity of a dilute solution of the compound of interest.

Table A.1H.9 HPLC Conditions for Enzyme Inhibitors

Compound Mobile phase Detector Retention time Limit ofdetection

AEBSF 40:60 (v/v)acetonitrile:0.1%trifluoroacetic acid

UV 225 nm 9.5 min 0.1 µg/ml

APMSF 40:60 (v/v)acetonitrile:0.1%trifluoroacetic acid

UV 232 nm 7.7 min 0.5 µg/ml

PMSF 50:50 (v/v)acetonitrile:water

UV 220 nm 8 min 0.9 µg/ml

TLCK 40:60 (v/v)acetonitrile:0.1%trifluoroacetic acid

UV 228 nm 9.5 min 0.37 µg/ml

TPCK 48:52 (v/v) acetonitrile:10mM pH 7 phosphate buffer

UV 228 nm 10.5 min 2 µg/ml


A.1H.30


REAGENTS AND SOLUTIONS

Use deionized, distilled water in all recipes and protocol steps. For common stock solutions, seeAPPENDIX 2; for suppliers, see APPENDIX 4.

Cyanide detection reagentStir 3.0 g barbituric acid in 10 ml water. Add 15 ml of 4-methylpyridine and 3 mlconcentrated HCl while continuing to stir. Cool and dilute to 50 ml with water. Storeat room temperature.


Decontamination solutionDissolve 4.2 g sodium nitrite (0.2 M final) and 20 ml hypophosphorous acid (3.3%w/v final) in 300 ml water. Prepare fresh.

Horseradish peroxidaseDissolve hydrogen-peroxide oxidoreductase (EC 1.11.1.7 [Type II]; specific activity150 to 200 purpurogallin U/mg, Sigma) in 1 g/liter sodium acetate to give 30 U/ml.Prepare fresh daily.

For small-scale reactions, a more dilute solution can be used to avoid working withinconveniently small volumes.

pH 5 buffer2.04 g potassium hydrogen phthalate (0.05 M final)38 ml 0.1 M potassium hydroxide (15 mM)H2O to 200 mlStore at room temperature

Phosphate buffer13.6 g monobasic potassium phosphate (KH2PO4; 0.1 M final)0.28 g dibasic sodium phosphate (Na2HPO4; 2 mM final)3.0 g potassium bromide (KBr; 25 mM final)1 liter H2OStore at room temperature

Potassium bromide is necessary to make the assay for cyanide work correctly.

Sodium azide indicator solution0.1 g bromocresol purple (0.4% final)18.5 ml 0.01 M potassium hydroxide (KOH; 7.4 mM final)H2O to 25 mlStore at room temperature

Sulfanilic acid solutionDissolve 0.5 g sulfanilic acid in 150 ml of 15% (v/v) aqueous acetic acid. Useimmediately.

LITERATURE CITED

Bretherick, L. (ed.) 1986. Hazards in the Chemical Laboratory, 4th ed. Royal Society of Chemistry, London.

Bretherick, L., Urben, P.G., and Pitt, M.J. 1999. Bretherick’s Handbook of Reactive Chemical Hazards, 6thed. Butterworth-Heinemann, London.

Castegnaro, M., Barek, J., Dennis, J., Ellen, G., Klibanov, M., Lafontaine, M., Mitchum, R., van Roosmalen,P., Sansone, E.B., Sternson, L.A., and Vahl, M. (eds.) 1985. Laboratory Decontamination and Destructionof Carcinogens in Laboratory Wastes: Some Aromatic Amines and 4-Nitrobiphenyl. IARC ScientificPublications No. 64. International Agency for Research on Cancer, Lyon, France.

Cunniff, P. (ed.) 1995. Official Methods of Analysis of the Association of Official Analytical Chemists, 16thed., Ch. 4, p. 14. Association of Official Analytical Chemists, Arlington, Va.


A.1H.31


Degenhardt-Langelaan, C.E.A.M. and Kientz, C.E. 1996. Capillary gas chromatographic analysis of nerveagents using large volume injections. J. Chromatogr. A.723:210-214.

Forsberg, K. and Keith, L.H. 1999. Chemical Protective Clothing Performance Index Book, 2nd ed. JohnWiley & Sons, New York.

Furr, A.K. (ed.) 2000. CRC Handbook of Laboratory Safety, 5th ed. CRC Press, Boca Raton, Fla.

Lewis, R.J. Sr. 1999. Sax’s Dangerous Properties of Industrial Materials, 10th ed. John Wiley & Sons, NewYork.

Lunn, G. and Sansone, E.B. 1985a. Destruction of cyanogen bromide and inorganic cyanides. Anal. Biochem.147:245-250.

Lunn, G. and Sansone, E.B. 1985b. Validation of techniques for the destruction of dimethyl sulfate. Am. Ind.Hyg. Assoc. J. 46:111-114.

Lunn, G. and Sansone, E.B. 1987. Ethidium bromide: Destruction and decontamination of solutions. Anal.Biochem. 162:453-458.

Lunn, G. and Sansone, E.B. 1989. Decontamination of ethidium bromide spills. Appl. Ind. Hyg. 4:234-237.

Lunn, G. and Sansone, E.B. 1990a. Validated methods for degrading hazardous chemicals: Some alkylatingagents and other compounds. J. Chem. Educ. 67:A249-A251.

Lunn, G. and Sansone, E.B. 1990b. Degradation of ethidium bromide in alcohols. BioTechniques 8:372-373.

Lunn, G. and Sansone, E.B. 1991a. The safe disposal of diaminobenzidine. Appl. Occup. Environ. Hyg.6:49-53.

Lunn, G. and Sansone, E.B. 1991b. Decontamination of aqueous solutions of biological stains. Biotech.Histochem. 66:307-315.

Lunn, G. and Sansone, E.B. 1991c. Decontamination of ethidium bromide spills-author’s response. Appl.Occup. Environ. Hyg. 6:644-645.

Lunn, G. and Sansone, E.B. 1994a. Destruction of Hazardous Chemicals in the Laboratory, 2nd ed. JohnWiley & Sons, New York.

Lunn, G. and Sansone, E.B. 1994b. Safe disposal of highly reactive chemicals. J. Chem. Educ. 71:972-976.

Lunn, G. and Sansone, E.B. 1994c. Degradation and disposal of some enzyme inhibitors. Scientific note.Appl. Biochem. Biotechnol. 48:57-59.

Lunn, G. and Sansone, E.B. 1994d. Safe disposal of diisopropyl fluorophosphate (DFP). Appl. Biochem.Biotechnol. 49:165-171.

Lunn, G., Klausmeyer, P.K., and Sansone, E.B. 1994. Removal of biological stains from aqueous solutionusing a flow-through decontamination procedure. Biotech. Histochem. 69:45-54.

Manufacturing Chemists Association. 1973. Laboratory Waste Disposal Manual. p. 136. ManufacturingChemists Association, Washington, D.C.

Mason, K.G. 1967. Hydrogen azide. In Mellor’s Comprehensive Treatise on Inorganic and TheoreticalChemistry, Vol. VIII (Suppl. II) pp. l-15. John Wiley & Sons, New York.

National Research Council. 1983. Prudent Practices for Disposal of Chemicals from Laboratories, p. 88.National Academy Press, Washington, D.C.

O’Neil, M.J. (ed.) 2001. The Merck Index, 13th ed. Merck & Co., Whitehouse Station, N.J.

Patnode, W. and Wilcock, D.F. 1946. Methylpolysiloxanes. J. Am. Chem. Soc. 68:358-363.

Shirakashi, T., Nakayama, K., Kakii, K., and Kuriyama, M. 1986. Removal of mercury from laboratory wastewater with iron powder. Chem. Abstr. 105:213690y.

Snyder, L.R., Kirkland, J.J., and Glajch, J.L. 1997. Practical HPLC Method Development, 2nd ed. John Wiley& Sons, New York.

KEY REFERENCES

The following are good general references for laboratory safety.

American Chemical Society, Committee on Chemical Safety. 1995. Safety in Academic Chemistry Labora-tories, 6th ed. American Chemical Society, Washington, D.C.

Castegnaro, M. and Sansone, E.B. 1986. Chemical Carcinogens. Springer-Verlag, New York.

DiBerardinis, L.J., First, M.W., Gatwood, G.T., and Seth, A.K. 2001. Guidelines for Laboratory Design,Health and Safety Considerations, 3rd ed. John Wiley & Sons, New York.

Fleming, D.D., Richardson, J.H., Tulis, J.J., and Vesley, D. 1995. Laboratory Safety, Principles and Practices,2nd ed. American Society for Microbiology, Washington, D.C.

Freeman, N.T. and Whitehead, J. 1982. Introduction to Safety in the Chemical Laboratory. Academic Press,San Diego.


A.1H.32


Fuscaldo, A.A., Erlick, B.J., and Hindman, B. (eds.) 1980. Laboratory Safety, Theory and Practice. AcademicPress, San Diego.

Lees, R. and Smith, A.F. (eds.) 1984. Design, Construction, and Refurbishment of Laboratories. EllisHorwood, Chichester, United Kingdom.

Montesano, R., Bartsch, H., Boyland, E., Della Porta, G., Fishbein, L., Griesemer, R.A., Swan, A.B., andTomatis, L. (eds.) 1979. Handling Chemical Carcinogens in the Laboratory, Problems of Safety. IARCScientific Publications No. 33. International Agency for Research on Cancer, Lyon, France.

National Research Council. 1995. Prudent Practices in the Laboratory: Handling and Disposal of Chemicals.National Academy Press, Washington, D.C.

Occupational Health and Safety. 1993. National Safety Council, Chicago.

Pal, S.B. (ed.) 1991. Handbook of Laboratory Health and Safety Measures, 2nd ed. Kluwer AcademicPublishers, Hingham, Mass.

Rosenlund, S.J. 1987. The Chemical Laboratory: Its Design and Operation: A Practical Guide for Plannersof Industrial, Medical, or Educational Facilities. Noyes Publishers, Park Ridge, N.J.

Young, J.A. (ed.) 1991. Improving Safety in the Chemical Laboratory: A Practical Guide, 2nd ed. John Wiley& Sons, New York.

INTERNET RESOURCES

http://www.ilpi.com/msds/index.html

Where to find MSDSs on the internet. Contains links to general sites, government and nonprofit sites, chemicalmanufacturers and suppliers, pesticides, and miscellaneous sites.

http://www.OSHA.gov

OSHA web site.

http://www.osha-slc.gov/OshStd_data/1910_1450.html

Text of OSHA Standard 29 CFR 1910.1450: Occupational Exposure to Hazardous Chemicals in Laboratories.

http://www.osha-slc.gov/OshStd_data/1910_1000_TABLE_Z-1.html

Tables of permissible exposure limits (PELs) for air contaminants.

http://www.osha-slc.gov/OshStd_data/1910_1000_TABLE_Z-2.html

Tables of PELs for toxic and hazardous substances.

http://hazard.com/msds/index.php

Main site for Vermont SIRI. One of the best general sites to start a search. Browse manufacturers alphabeti-cally (for sheets not in the SIRI collection) or do a keyword search in the SIRI MSDS database. Lots ofadditional safety links and information.

http://siri.uvm.edu/msds

Alternate site for Vermont SIRI.

http://tis.eh.doe.gov/docs/osh_tr/ch5.html

DOE OSH technical reference chapter on personal protective equipment.

Contributed by George LunnBaltimore, Maryland

Gretchen LawlerPurdue UniversityWest Lafayette, Indiana


A.1H.33


APPENDIX 1ICommonly Used DetergentsDetergents are polar lipids that are soluble in water. The presence of both a hydrophobicand hydrophilic portion makes these compounds very useful for lysis of lipid membranes,solubilization of antigens, and washing of immune complexes.

TYPES OF DETERGENTS

A large variety of detergents are available (Helenius et al., 1979). For biochemical studies,they are usually categorized according to the type of hydrophilic group they contain—anionic, cationic, amphoteric, or nonionic. Tables A.1I.1 and A.1I.2 list commonly usedmembers of each type. In general, nonionic and amphoteric detergents are less denaturingfor proteins than ionic detergents. Sodium cholate and sodium deoxycholate are the leastdenaturing of the commonly used ionic detergents.

Two properties of detergents are important in their consideration for biological studies:the critical micelle concentration (CMC) and the micelle molecular weight (Table A.1I.1).The CMC is the concentration at which monomers of detergent molecules combine toform micelles; each detergent micelle has a characteristic micelle molecular weight.Detergents with a high micelle molecular weight, such as nonionic detergents, are difficultto remove from samples by dialysis. The CMC and the micelle molecular weight willvary depending on the buffer, salt concentration, pH, and temperature. In general, addingsalt will lower the CMC and raise the micelle size.

Table A.1I.1 Physical Properties of Commonly Used Detergentsa,b

Detergent mp (°C)Molecular weight (Da) CMC

Monomer Micelle % (w/v) M

Anionic

SDS 206 288 18,000 0.23 8.0 × 10−3

Cholate 201 430 4,300 0.60 1.4 × 10−2

Deoxycholate 175 432 4,200 0.21 5.0 × 10−3

Cationic

C16TAB 230 365 62,000 0.04 1.0 × 10−3

Amphoteric

LysoPC — 495 92,000 0.0004 7.0 × 10−6

CHAPS 157 615 6,150 0.49 1.4 × 10−3

Zwittergent 3-14 — 364 30,000 0.011 3.0 × 10−4

Nonionic

Octyl glucoside 105 292 8,000 0.73 2.3 × 10−2

Digitonin 235 1,229 70,000 — —

C12E8 — 542 65,000 0.005 8.7 × 10−5

Lubrol PX — 582 64,000 0.006 1.0 × 10−4

Triton X-100 — 650 90,000 0.021 3.0 × 10−4

Nonidet P-40 — 603 90,000 0.017 3.0 × 10−4

Tween 80 — 1,310 76,000 0.002 1.2 × 10−5

aReprinted with permission from IRL Press (see Jones et al., 1987).bAbbreviations: C16TAB, hexadecyl trimethylammonium bromide; CMC, critical micelle concentration;LysoPC, lysophosphatidylcholine; mp, melting point; SDS, sodium dodecyl sulfate.

Supplement 43

Contributed by John E. ColiganCurrent Protocols in Molecular Biology (1998) A.1I.1-A.1I.3Copyright © 1998 by John Wiley & Sons, Inc.

A.1I.1


CHOICE OF DETERGENTS

Ionic detergents are very good solubilizing agents, but they tend to denature proteins bydestroying native three-dimensional structures. This denaturing ability is useful forSDS-PAGE (UNIT 10.2), but is detrimental where native structure is important, as whenfunctional activities must be retained (antibody activity is usually retained in <0.1% SDS).Nonionic and mildly ionic detergents are less denaturing and can often be used tosolubilize membrane proteins while retaining protein-protein interactions. The followingdetergent properties are detrimental in certain procedures:

1. Phenol-containing detergents (e.g., Triton X-100 and NP-40) have a high absorbanceat 280 nm and hence interfere with protein monitoring during chromatography (mostionic detergents do not absorb at 280 nm; Brij- and Lubrol-series detergents arenonionic detergents that do not have substantial absorbance at 280 nm). Phenol-con-taining detergents also induce precipitation in the Folin protein assay (but they canbe used with the Bradford protein assay; UNIT 10.1A). Finally, they are readily iodinatedand so should not be present during radioiodination.

2. Many detergents have a very high micelle molecular weight (Table A.1I.1), whichmakes their use in gel filtration impossible since protein sizes are insignificant relativeto the micelle size. In addition, such detergents cannot be readily removed by dialysis.

3. Sodium cholate and sodium deoxycholate are insoluble below pH 7.5 or above anionic strength of 0.1%. SDS will often crystallize below 20°C.

4. Ionic detergents interfere with nondenaturing electrophoresis and isoelectric focus-ing.

Table A.1I.2 Chemical Properties of Commonly Used Detergentsa,b

PropertyIonic detergents Nonionic detergents

SDS CHO DOC C16 LYS CHA ZWI OGL DIG C12 LUB TNX NP-40 T80

Strongly denaturingc + − − + +/− − +/− − − − − − − −Dialyzable + + + + − + +/− + − − − − − −Ion exchangeabled + + + + − − − − − − − − − −Complexes ions + + + − − − − − − +/− +/− +/− +/− +/−Strong A280 − − − − − − − − − − − + + −Assay interference − − − − − − − − − − +/− +/− +/− +/−Cold precipitates + − + + − − − − − − − − − −High cost − − − − + + + + + + − − − −Availability + + + + + + +/− + + +/− + + + +Toxicity − − − − − − − − − − − − − −Ease of purification + + + + +/− + + − + − − − − −Radiolabeled + + + − + − − + − + + + + +Defined composition + + + + + + + + − − − − −Auto-oxidation − − − − − − − − + + + + +aAdapted from IRL Press (see Jones et al., 1987).bAbbreviations: C12, C12E8; C16, hexadecyl trimethylammonium bromide; CHA, CHAPS; CHO, cholate; DIG, digitonin;DOC, deoxycholate; LUB, lubrol PX; LYS, lysophosphatidylcholine; NP-40, Nonidet P-40; OGL, octyl glucoside; SDS,sodium dodecyl sulfate; T80, Tween 80; TNX, Triton X-100; ZWI, Zwittergent 3-14.c Denaturing refers to disruption of secondary and tertiary protein structure.dIonic detergents are unsuitable for ion-exchange chromatography (UNIT 10.10).

Detergents


A.1I.2

Detergents can be removed or exchanged for other detergents by a variety of procedures(Hjelmeland, 1979; Furth et al., 1984; Harlow and Lane, 1988). Ionic and amphotericdetergents can usually be removed by dialysis (APPENDIX 3C). Pierce makes Extracti-GelD for removing a variety of detergents from protein solutions (Pierce ImmunotechnologyCatalog and Handbook on Protein Modification).

LITERATURE CITED

Furth, A.J., Bolton, H., Potter, J., and Priddle, J.D. 1984. Separating detergents from proteins. MethodsEnzymol. 104:318-328.

Harlow, E. and Lane, D. 1988. Detergents. In Antibodies: A Laboratory Manual, pp. 687-689. Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.

Helenius, A., McCaslin, D.R., Fries, E., and Tanford, C. 1979. Properties of detergents. Methods Enzymol.56:734-749.

Hjelmand, L.M. 1979. Removal of detergents from membrane proteins. Methods Enzymol. 182:277-282.

Jones, O.T., Earnest, J.P. and McNamee, M.G. 1987. Solubilization and reconstitution of membrane proteins.In Biological Membranes: A Practical Approach (J. Findlay and W.H. Evans, eds.), pp. 142-143. IRLPress, Oxford.

KEY REFERENCES

Harlow and Lane, 1988. See above.

Provides properties of commonly used detergents and means of removing them from proteins.

Hjelmeland, L.M. and Chrambach, A. 1984. Solubilization of functional membrane proteins. MethodsEnzymol. 182:305-318.

Describes properties of detergents and how to use them to solubilize proteins.

Johnstone, A. and Thorpe, R. 1982. Isolation and fractionation of lymphocytes. In Immunochemistry inPractice, pp. 94-101. Blackwell Scientific, Oxford.

Provides details in use of detergents to solubilize cells and membranes.

Neugebaur, J.M. 1990. Detergents: An overview. Methods Enzymol. 182:239-282.

Provides details on detergent properties and how to choose one for a particular application.

Contributed by John E. ColiganNational Institute of Allergy and Infectious DiseasesBethesda, Maryland


A.1I.3


APPENDIX 1JCommon Conversion Factors

Table A.1J.1 lists some of the more common conversion factors for units of measure usedthroughout Current Protocols manuals, while Table A.1J.2 gives prefixes indicatingpowers of ten for SI units.

Table A.1J.3 gives conversions between temperatures on the Celsius (Centigrade) andFahrenheit scales. Celsius temperatures are converted to Fahrenheit temperatures bymultiplying the Celsius figure by 9, dividing by 5, and adding 32, or by multiplying theCelsius figure by 1.8 and adding 32. Fahrenheit is converted to Celsius by subtracting 32from the Fahrenheit figure, multiplying by 5, and dividing by 9. In Table A.1J.3, the centerfigure represents the temperature one has read on one of the scales; the figure to the leftis the conversion of that figure into Celsius if read in Fahrenheit, while that to the rightrepresents the conversion to Fahrenheit if read in Celsius: e.g., the temperature 88Fahrenheit converts to 31.1°C, while the temperature 88 Celsius converts to 190.4°F.

Supplement 49

Current Protocols in Molecular Biology (2000) A.1J.1-A.1J.8Copyright © 2000 by John Wiley & Sons, Inc.

Table A.1J.1 Unit of Measurement Conversion Chart

To convert: Into: Use the multiplier:

amperes per square centimeter (amp/cm2) amperes per square inch (amp/in.2) 6.452amperes per square meter (amp/m2) 104

amperes per square inch (amp/in.2) amperes per square centimeter (amp/cm2) 0.1550amperes per square meter (amp/m2) 1.55 × 103

ampere-hours (amp-hr) coulombs (C) 3.6 × 103

faradays 3.731 × 10−2

atmospheres (atm) bar 1.01325millimeters of mercury (mmHg) or torr 760tons per square foot (tons/ft2) 1.058

bar atmospheres (atm) 0.9869dynes per square centimeter (dyn/cm2) 106

kilograms per square meter (kg/m2) 1.020 × 104

pounds per square foot (lb/ft2) 2,089pounds per square inch (lb/in.2 or psi) 14.50

British thermal units (Btu) ergs 1.0550 × 1010

gram-calories (g-cal) 252.0horsepower-hours (hp-hr) 3.931 × 104

joules (J) 1,054.8kilogram-calories (kg-cal) 0.2520kilogram-meters (kg-m) 107.5kilowatt-hours (kW-hr) 2.928 × 10−4

British thermal unit per minute (Btu/min) foot-pounds per second (ft-lb/sec) 12.96horsepower (hp) 2.356 × 10−2

watts (W) 17.57

bushels cubic feet (ft3) 1.2445cubic inches (in.3) 2,150.4cubic meters (m3) 3.524 × 10−2

liters 35.24quarts, dry 32.0

continued

A.1J.1

degrees Celsius or Centigrade (°C) degrees Fahrenheit (°F) (°C × 9⁄5) + 32Kelvin (K) (°C) + 273.15

degree Fahrenheit (°F) degrees Celsius (°C) 5⁄9 × (°F − 32)Kelvin (K) [5⁄9 × (°F − 32)] + 273.15

centimeters (cm) feet (ft) 3.281 × 10−2

inches (in.) 0.3937kilometers (km) 10−5

meters (m) 10−2

miles 6.214 × 10−6

millimeters (mm) 10.0mils 393.7yards 1.094 × 10−2

centimeters per second (cm/sec) feet per minute (ft/min) 1.1969feet per second (ft/sec) 3.281 × 10−2

kilometers per hour (km/hr) 3.6 × 10−2

meters per minute (m/min) 0.6miles per hour (miles/hr) 2.237 × 10−2

miles per minute (miles/min) 3.728 × 10−4

coulombs (C) faradays 1.036 × 10−5

coulombs per square centimeter (C/cm2) coulombs per square inch (C/in.2) 64.52coulombs per square meter (C/m2) 104

coulombs per square inch (C/in.2) coulombs per square centimeter (C/cm2) 0.1550coulombs per square meter (C/m2) 1.55 × 103

cubic centimeters (cm3) cubic feet (ft3) 3.531 × 10−5

cubic inches (in.3) 6.102 × 10−2

cubic meters (m3) 10−6

cubic yards 1.308 × 10−6

gallons, U.S. liquid 2.642 × 10−4

liters 10−3

pints, U.S. liquid 2.113 ×10−3

quarts, U.S. liquid 1.057 × 10−3

days hours (hr) 24.0minutes (min) 1.44 × 103

seconds (sec) 8.64 × 104

degrees (of angle; °) minutes (min) 60.0quadrants, of angle 1.111 × 10−2

radians (rad) 1.745 × 10−2

seconds (sec) 3.6 × 104

drams grams (g) 1.7718grains 27.3437ounces, avoirdupois (oz) 6.25 × 10−2

dynes (dyn) joules per centimeter (J/cm) 10−7

joules per meter (J/m) or newtons (N) 10−5

kilograms (kg) 1.020 × 10−6

pounds (lb) 2.248 × 10−6

continued

Table A.1J.1 Unit of Measurement Conversion Chart, continued



A.1J.2

faradays ampere-hours (amp-hr) 26.80coulombs (C) 9.649 × 10−4

foot-pounds per minute (ft-lb/min) British thermal units per minute (Btu/min) 1.286 × 10−3

foot-pounds per second (ft-lb/sec) 1.667 × 10−2

horsepower (hp) 3.030 × 10−5

kilogram-calories per minute (kg-cal/min) 3.24 × 10−4

kilowatts (kW) 2.260 × 10−5

grams (g) decigrams (dg) 10dekagrams (Dg) 0.1dynes (dyn) 980.7grains 15.43hectograms (hg) 10−2

kilograms (kg) 10−3

micrograms (µg) 106

milligrams (mg) 103

ounces, avoirdupois (oz) 3.527 × 10−2

ounces, troy 3.215 × 10−2

pounds (lb) 2.205 × 10−3

horsepower (hp) horsepower, metric 1.014

inches (in.) centimeters (cm) 2.540feet (ft) 8.333 × 10−2

meters (m) 2.540 × 10−2

miles 1.578 × 10−5

millimeters (mm) 25.40yards 2.778 × 10−2

inches of mercury (in. Hg) atmospheres (atm) 3.342 × 10−2

kilogram per square centimeter (kg/cm2) 3.453 × 10−2

kilograms per square meter (kg/m2) 345.3pounds per square foot (lb/ft2) 70.73pounds per square inch (lb/in.2 or psi) 0.4912

joules (J) British thermal units (Btu) 9.480 × 10−4

ergs 107

foot-pounds (ft-lb) 0.7376kilogram-calories (kg-cal) 2.389 × 10−4

kilogram-meters (kg-m) 0.1020newton-meter (N-m) 1watt-hours (W-hr) 2.778 × 10−4

Kelvin (K) degrees Celsius (°C) K − 273.13degrees Fahrenheit (°F) [(K − 273.13) × 9⁄5] + 32

kilolines maxwells (Mx) 103

kilometers (km) centimeters (cm) 105

feet (ft) 3,281inches (in.) 3.937 × 104

meters (m) 103

miles 0.6214yards 1,094

continued




A.1J.3

kilowatts (kW) British thermal units per minute (Btu/min) 56.92foot-pounds per minute (ft-lb/min) 4.426 × 104

horsepower (hp) 1.341kilogram-calories per minute (kg-cal/min) 14.34

liters bushels, U.S. dry 2.838 × 10−2

cubic centimeters (cm3) 103

cubic feet (ft3) 3.531 × 10−2

cubic inches (in.3) 61.02cubic meters (m3) 10−3


gallons, U.S. liquid 0.2642gallons, imperial 0.21997kiloliter (kl) 10−3

pints, U.S. liquid 2.113quarts, U.S. liquid 1.057

maxwells (Mx) webers (W) 10−8

micrograms (µg) grams (g) 10−6

microliters (µl) liters 10−6

milligrams (mg) grams (g) 10−3

milligrams per liter (mg/liter) parts per million (ppm) 1.0

millihenries (mH) henries (H) 10−3

milliliters (ml) liters 10−3

millimeters (mm) centimeters (cm) 0.1feet (ft) 3.281 × 10−3

inches (in.) 3.937 × 10−2

kilometers (km) 10−6

meters (m) 10−3

miles 6.214 × 10−7

millimeters of mercury (mmHg) or torr atmospheres (atm) 1.316 × 10−3

kilograms per square meter (kg/m2) 136.0pounds per square foot (lb/ft2) 27.85pounds per square inch (lb/in.2 or psi) 0.1934

nepers (Np) decibels (dB) 8.686

newtons (N) dynes (dyn) 105

kilograms, force (kg) 0.10197162pounds, force (lb) 4.6246 × 10−2

ohms (Ω) megaohms (MΩ) 106

microhms (µΩ) 10−6

ounces, avoirdupois drams 16.0grains 437.5grams (g) 28.349527pounds (lb) 6.25 × 10−2

ounces, troy 0.9115tons, metric 2.835 × 10−5

continued




A.1J.4

ounces, fluid cubic inches (in3) 1.805liters 2.957 × 10−2

ounces, troy grains 480.0grams (g) 31.103481ounces, avoirdupois (oz) 1.09714pounds, troy 8.333 × 10−2

pascal (P) newton per square meter (N/m2) 1

pounds, force (lb) newtons (N) 21.6237

pounds per square foot (lb/ft2) atmospheres (atm) 4.725 × 10−4

inches of mercury (in. Hg) 1.414 × 10−2

kilograms per square meter (kg/m2) 4.882pounds per square inch (lb/in2 or psi) 6.944 × 10−3

pounds per square inch (lb/in.2 or psi) atmospheres (atm) 6.804 × 10−2

inches of mercury (in. Hg) 2.036kilograms per square meter (kg/m2) 703.1pounds per square foot (lb/ft2) 144.0bar 6.8966 × 10−2

quadrants, of angle degrees (°) 90.0minutes (min) 5.4 × 103

radians (rad) 1.571seconds (sec) 3.24 × 105

quarts, dry cubic inches (in.3) 67.20

quarts, liquid cubic centimeters (cm3) 946.4cubic feet (ft3) 3.342 × 10−2

cubic inches (in.3) 57.75cubic meters (m3) 9.464 × 10−4


gallons 0.25liters 0.9463

radians (rad) degrees (°) 57.30minutes (min) 3,438quadrants 0.6366seconds (sec) 2.063 × 105

torr see millimeter of mercury

watts (W) British thermal units per hour (Btu/hr) 3.413British thermal units per min (Btu/min) 5.688 × 10−2

ergs per second (ergs/sec) 107

webers (Wb) maxwells (M) 108

kilolines 105




A.1J.5

Useful Data

Table A.1J.2 Power of Ten Prefixes for SI Units

Prefix Factor Abbreviation

atto 10−18 afemto 10−15 fpico 10−12 pnano 10−9 nmicro 10−6 µmilli 10−3 mcenti 10−2 cdeci 10−1 ddeca 101 dahecto 102 hkilo 103 kmyria 104 mymega 106 Mgiga 109 Gtera 1012 Tpeta 1015 Pexa 1018 E


A.1J.6

CommonConversion

Factors

Table A.1J.3 Celsius/Fahrenheit Temperature Conversion Chart

Degrees Celsius (°C) Temperature Degrees Fahrenheit (°F)

−17.8 0 32.0−17.2 1 33.8−16.7 2 35.6−16.1 3 37.4−15.6 4 39.2−15.0 5 41.0−14.4 6 42.8−13.9 7 44.6−13.3 8 46.4−12.8 9 48.2−12.2 10 50.0−11.7 11 51.8−11.1 12 53.6−10.6 13 55.4−10.0 14 57.2−9.4 15 59.0−8.9 16 60.8−8.3 17 62.6−7.8 18 64.4−7.2 19 66.2−6.7 20 68.0−6.1 21 69.8−5.6 22 71.6−5.0 23 73.4−4.4 24 75.2−3.9 25 77.0−3.3 26 78.8−2.8 27 80.6−2.2 28 82.4−1.7 29 84.2−1.1 30 86.0−0.6 31 87.80.0 32 89.60.6 33 91.41.1 34 93.21.7 35 95.02.2 36 96.82.8 37 98.63.3 38 100.43.9 39 102.24.4 40 104.05.0 41 105.85.6 42 107.66.1 43 109.46.7 44 111.27.2 45 113.07.8 46 114.88.3 47 116.68.9 48 118.49.4 49 120.2

10.0 50 122.010.6 51 123.8

continued


A.1J.7

Useful Data

11.1 52 125.611.7 53 127.412.2 54 129.212.8 55 131.013.3 56 132.813.9 57 134.614.4 58 136.415.0 59 138.215.6 60 140.016.1 61 141.816.7 62 143.617.2 63 145.417.8 64 147.218.3 65 149.018.9 66 150.819.4 67 152.620.0 68 154.420.6 69 156.221.1 70 158.021.7 71 159.822.2 72 161.622.8 73 163.423.3 74 165.223.9 75 167.024.4 76 168.825.0 77 170.625.6 78 172.426.1 79 174.226.7 80 176.027.2 81 177.827.8 82 179.628.3 83 181.428.9 84 183.229.4 85 185.030.0 86 186.830.6 87 188.631.1 88 190.431.7 89 192.232.2 90 194.032.8 91 195.833.3 92 197.633.9 93 199.434.4 94 201.235.0 95 203.035.6 96 204.836.1 97 206.836.7 98 208.437.2 99 210.237.8 100 212.0

Table A.1J.3 Celsius/Fahrenheit Temperature Conversion Chart,continued

Degrees Celsius (°C) Temperature Degrees Fahrenheit (°F)


A.1J.8

CommonConversion

Factors

APPENDIX 1KCompendium of Drugs Commonly Used inMolecular Biology ResearchThe following appendix includes an alphabetical list of drugs commonly used to examinevarious biological processes. Table A.1K.1 lists the drugs by activity and provides recentreferences. Indicated under each drug listed is its mode of action, generally includingseveral specific experimental examples; solvent(s) used to solubilize the drug; stock andworking concentrations or ranges; storage conditions; and duration of incubation withcells to achieve the desired effects. Except where indicated, the majority of drugs in thislist are cell-permeant. However, despite the well characterized selectivity of many of thefollowing drugs in vitro, the corresponding effects upon their intracellular targets maynot be precisely determined directly by their extracellular concentrations, since theircell-permeation properties are not known. Therefore, several different concentrations ofany particular drug, as well as alternative methods of determining drug selectivity, shouldbe examined.

Several of these drugs are members of large families, such as those targeting proteinkinases and phosphatases, as well as those that affect intracellular Ca2+ levels. Many ofthese family members have different selectivities and potencies toward similar targets,and a complete listing is not included here. The reader may consult catalogs from thefollowing companies, which have several of these family members available: Sigma,Alexis Biochemicals (including LC Laboratories), Calbiochem, Biomol, MolecularProbes, Boehringer Mannheim, Oxford Glycosystems, and Avanti Polar Lipids.

Although not specifically indicated, many of the following drugs are hazardous andshould be handled with extreme care. Material Safety Data Sheets (MSDSs) are oftenprovided for products that are hazardous or toxic. In some cases, these products are newand have not been tested for toxicity. Thus, care should be taken to ensure the safe handlingof all products.

Supplement 59

Contributed by Nelson B. ColeCurrent Protocols in Molecular Biology (2002) A1.K.1-A1.K.26Copyright © 2002 by John Wiley & Sons, Inc.

Table A.1K.1 Biological Activity of Drugs Commonly Used In Molecular Biological Research

Drug References

DNA replication inhibitorsAphidicolin Borner et al., 1995. Cancer Res. 55:2122-2128; Debec et al., 1996. J.

Cell Biol. 134:103-115; Jackson et al., 1995. J. Cell Biol. 130:755-769; Urbani et al., 1995. Exp. Cell Res. 219:159-168

Ara-C Grant et al., 1994. Oncol. Res. 6:87-99; Tomkins et al., 1994. J. CellSci. 107:1499-1507

Camptothecin Carettoni et al., 1994. Biochem. J. 299:623-629; Desai et al., 1997.JBC 272:24159-24164; Li et al., 1994. JBC 269:7051-7054

Etoposide Kaufmann et al., 1993. Cancer Res. 53:3976-3985; Meyer et al., 1997.J. Cell Biol. 136:775-788; Shao et al., 1997. JBC 272:31321-31325;Terada et al., 1993. J. Med. Chem. 36:1689-1699; Wozniak et al.,1991. J. Clin. Oncol. 9:70-76

Hydroxyurea Anand et al., 1995. Cancer Lett. 88:101-105; Nishijima et al., 1997. J.Cell Biol. 138:1105-1116; Oliver et al., 1997. JBC 272:10624-10630

L-Mimosine Gilbert et al., 1995. JBC 270:9597-9606; Lin et al., 1996. JBC271:2548-2556; Park et al., 1997. J. Neurosci. 17:1256-1270

continued

A.1K.1


Drugs affecting the cytoskeletonColcemid Barlow et al., 1994. J. Cell Biol. 126:1017-1029; Bonfoco et al., 1995.

Exp. Cell Res. 218:189-200; Goswami et al., 1994. Exp. Cell Res.214:198-208

Colchicine Bonfoco et al., 1995. Exp. Cell Res. 218:189-200; Lindenboim et al.,1995. J. Neurochem. 64:1054-1063

Cytochalasin B Benya and Padilla, 1993. Exp. Cell Res. 204:268-277; Takeshita et al.,1998. Cancer Lett. 126:75-81; Tanaka et al., 1994. Exp. Cell Res.213:242-252

Cytochalasin D Cooper, 1987. J. Cell Biol. 105:1473-1478; Radhakrishna andDonaldson, 1997. J. Cell Biol. 139:49-61; Sasaki et al., 1995. PNAS92:2026-2030; Wang et al., 1994. Am. J. Physiol. 267:F592-F598

Latrunculins Wada et al., 1998. J. Biochem. 123:946-952; Lamaze et al., 1997. JBC272:20332-20335; Spector et al., 1989. Cell Motil. Cytoskeleton13:127-144

Nocodazole Cole et al., 1996. Mol. Biol Cell. 7:631-650; Liao et al., 1995. J. CellSci. 108:3473-3483; Vasquez et al., 1997. Mol. Biol. Cell 8:973-985

Taxol Derry et al., 1995. Biochemistry 34:2203-2211; Ding et al., 1990.Science 248:370-372; Gallo, 1998. J. Neurobiol. 35:121-140; Jordanet al., 1993. PNAS 90:9552-9556; Mogensen and Tucker, 1990. J. CellSci. 97:101-107

Vinblastine Dhamodharan et al., 1995. Mol. Biol. Cell 6:1215-1229; Lobert et al.,1998. Cell Motil. Cytoskeleton 39:107-121; Panda et al., 1996. JBC271:29807-29812; Rai and Wolff, 1996. JBC 271:14707-14711; Takiet al., 1998. J. Neurooncol. 36:41-53; Tsukidate et al., 1993. Am. J.Pathol. 143:918-925

Drugs affecting intracellular Ca2+

A23187 Elia et al., 1996. JBC 27:16111-16118; Kao, 1994. Methods Cell Biol.40:155-181; Kao et al., 1990. J. Cell Biol. 111:183-196

BAPTA Bissonnette et al., 1994. Am. J. Physiol. 267:G465-G475; Smith et al.,1992. Biochem. J. 288:925-929; Tsien, 1980. Biochemistry 19:2396-2404

Ionomycin Aagaard-Tillery and Jelinek, 1995. J. Immunol. 155:3297-3307; Rocket al., 1997. JBC 272:33377-33383; Stewart et al., 1998. J. Cell Biol.140:699-707

Thapsigargin Kuznetsov et al., 1993. JBC 268:2001-2008; Lodish et al., 1992. JBC267:12753-12760; Takemura et al., 1989. JBC 264:12266-12271; Wonand Orth, 1995. Endocrinology 136:5399-5408; Wong et al., 1993.Biochem. J. 289:71-79

Drugs affecting oligosaccharide biosynthesis/processingCastanospermine Ahmed et al., 1995. Biochem. Biophys. Res. Commun. 208:267-273;

Bass et al., 1998. J. Cell Biol. 141:637-646; Gruters et al., 1987.Nature 330:74-77; Martina et al., 1998. JBC 273:3725-3731

Deoxymannojirimycin Elbein et al., 1984. Arch. Biochem. Biophys. 235:579-588; McDowellet al., 1987. Virology 161:37-44; Slusarewicz and Warren, 1995.Glycobiology 5:154-155; Wojczyk et al., 1998. Glycobiology 8:121-130

Deoxynojirimycin Labriola et al., 1995. J. Cell Biol. 130:771-779; Martina et al., 1998.JBC 273:3725-3731; Romero et al., 1985. Biochem. J. 226:733-740;Schlesinger et al., 1984. JBC 259:7597-7601

Tunicamycin Bush et al., 1994. Biochem. J. 303:705-708; Kuznetsov et al., 1997.JBC 272:3057-3063; Lodish and Kong, 1984. J. Cell Biol. 98:1720-1729

Table A.1K.1 Biological Activity of Drugs Commonly Used In Molecular Biological Research,continued

Drug References

continued


A.1K.2

Compendium ofDrugs Commonly

Used inMolecular

Biology Research

Drugs affecting the pH of intracellular organellesAmmonium chloride Breuer et al., 1995. JBC 270:24209-24215; Davis and Mecham, 1996.

JBC 271:3787-3794; Smith et al., 1997. JBC 272:5640-5646Bafilomycin A1 Bowman et al., 1988. PNAS 85:7972-7976; Calvert and Sanders, 1995.

JBC 270:7272-7280; Furuchi et al., 1993. JBC 268:27345-27348CCCP Arai et al., 1996. Biochem. Biophys. Res. Commun. 227:433-439;

Babcock et al., 1997. J. Cell Biol. 136:833-844; Kao, 1994. MethodsCell Biol. 40:155-181; Simpson and Russell, 1996. JBC 271:33493-33501

Chloroquine Claus et al., 1998. JBC 273:9842-9851; Garcia-Sainz and Mendoza-Mendoza, 1998. Eur. J. Pharmacol. 342:333-338; Passos and Garcia,1998. Biochem. Biophys. Res. Commun. 245:155-160; Wunsch et al.,1998. J. Cell Biol. 140:335-345

Concanamycin B Akifusa et al., 1998. Exp. Cell Res. 238:82-89; Nishihara et al., 1995.Biochem. Biophys. Res. Commun. 212:255-262;Yilla et al., 1993. JBC 268:19092-19100

Monensin Griffiths et al., 1983. J. Cell Biol. 96:835-850; Hardy et al., 1997. JBC272:6812-6817; Kallen et al., 1993. Biochim. Biophys. Acta 1166:305-308; Shiao and Vance, 1993. JBC 268:26085-26092

Nigericin Sandvig et al., 1989. Methods Cell Biol. 32:365-382; Scorrano et al.,1997. JBC 272:12295-12299; Vercesi et al., 1993. JBC 268:8564-8568

Drugs that lead to increased intracellular cAMP levels8-Bromo–cyclic AMP Boyer and Thiery, 1993. J. Cell Biol. 120:767-776; Hei et al., 1991.

Mol. Pharmacol. 39:233-238; Sandberg et al., 1991. Biochem. J.279:521-527

Cholera toxin Hansen and Casanova, 1994. J. Cell Biol. 126:677-687; Lencer et al.,1995. J. Cell Biol. 131:951-962; Ma and Weiss, 1995. Methods CellBiol. 49:471-485; Moss and Vaughan, 1992. Curr. Top. Cell. Regul.32:49-72

Dibutyryl cyclic AMP Cong et al., 1998. JBC 273:660-666; O’Malley et al., 1997. J. CellBiol. 138:159-165; Sandvig et al., 1994. J. Cell Biol. 126:53-64; Yuanet al., 1996. JBC 271:27090-27098

Forskolin Galli et al., 1995. J. Neurosci. 15:1172-1179; Lippincott-Schwartz etal., 1991. J. Cell Biol. 112:567-577; Laurenza et al., 1989. TrendsPharmacol. Sci. 10:442-447; Nickel et al., 1996. JBC 271:15870-15873

Kinase inhibitorsBisindolylmaleimide I(GF 109203 X)

Das and White, 1997. JBC 272:14914-14920; Toullec et al., 1991.JBC 266:15771-15781; Uberall et al., 1997. JBC 272:4072-4078

Calphostin C Dubyak and Kertesy, 1997. Arch. Biochem. Biophys. 341:129-139;Hartzell and Rinderknecht, 1996. Am. J Physiol. 270:C1293-C1299;Jarvis et al., 1994. Cancer Res. 54:1707-1714

Chelerythrine chloride Barg et al., 1992. J. Neurochem. 59:1145-1152; Herbert et al., 1990.Biochem. Biophys. Res. Commun. 172:993-999; Jarvis et al., 1994.Cancer Res. 54:1707-1714; Jia et al., 1997. JBC 272:4978-4984;Kandasamy et al., 1995. JBC 270:29209-29216

Genistein Akiyama et al., 1987. JBC 262:5592-5595; Chen et al., 1997. JBC272:27401-27410; Kranenburg et al., 1997. J. Cell Sci. 110:2417-2427

H-7 Barria et al., 1997. Science 276:2042-2045; Kawamoto and Hidaka,1984. Biochem. Biophys. Res. Commun. 125:258-264; Wang et al.,1997. JBC 272:1817-1821

continued


Drug References


A.1K.3


Herbimycin A Cowen et al., 1996. JBC 271:22297-22300; Fukazawa et al., 1991.Biochem. Pharmacol. 42:1661-1671; Tiruppathi et al., 1997. JBC272:25968-25975

KN-62 Bouvard et al., 1998. J. Cell Sci. 111:657-665; Enslen and Soderling,1994. JBC 269:20872-20877; Wang Hy et al., 1997. JBC 272:1817-1821; Doroudchi et al., 1997. J. Neurosci. Res. 50:514-521.

LY294002 Vlahos et al., 1994. JBC 269:5241-5248; Vlahos et al., 1995. J.Immunol. 154:2413-2422

ML-7 Ohkubo et al., 1996. Eur. J. Pharmacol. 298:175-183; Saitoh et al.,1987. JBC 262:7796-7801; Watanabe et al., 1998. FASEB J. 12:341-348

Olomoucine Abraham et al., 1995. Biol. Cell 83:105-120; Glab et al., 1994. FEBSLett. 353:207-211; Howell et al., 1997. Cell. Motil. Cytoskeleton38:201-214; Misteli and Warren, 1995. J. Cell Sci. 108:2715-2727

PD 98059 Acharya et al., 1998. Cell 92:183-192; Alessi et al., 1995. JBC270:27489-27494; Dudley et al., 1995. PNAS 92:7686-7689; Karpovaet al., 1997. Am. J. Physiol. 272:L558-L565; Waters et al., 1995. JBC270:20883-20886

Piceatannol Keely and Parise, 1996. JBC 271:26668-26676; Oliver et al., 1994.JBC 269:29697-29703; Peters et al., 1996. JBC 271:4755-4762

Staurosporine Couldwell et al., 1994. FEBS Lett. 345: 43-46; Kiss and Deli, 1992.Biochem. J. 288: 853-858; Janicke et al., 1998. JBC 273:9357-9360;Orr et al., 1998. JBC 273:3803-3807

Tyrphostins Antonyak et al., 1998. JBC 273:2817-2822; Austin and Shields, 1996.J. Cell Biol. 135:1471-1483; Gazit et al., 1989. J. Med. Chem.32:2344-2352; Gohla et al., 1998. JBC 273:4653-4659

Wortmannin Cross et al., 1995. JBC 270:25352-25355; Goeger et al., 1988.Biochem. Pharmacol. 37:978-981; Jones and Howell, 1997. J. CellBiol. 139:339-349; Ptasznik et al., 1997. J. Cell Biol. 137:1127-1136

Phosphatase inhibitorsCalyculin A Ishihara et al., 1989. Biochem. Biophys. Res. Commun. 159:871-877;

Murakami et al., 1994. Neurosci. Lett. 176: 181-184; Takeuchi et al.,1994. Biochem. Biophys. Res. Commun. 205:1803-1807

Microcystin-LR Bagu et al., 1997. JBC 272:5087-5097; Eriksson et al., 1990. Biochim.Biophys. Acta 1025:60-66; Honkanen et al., 1990. JBC 265:19401-19404; Rabouille et al., 1995. J. Cell Biol. 129:605-618; Toivola et al.,1997. J. Cell Sci. 110:23-33

Okadaic acid Gjertsen et al., 1994. J. Cell Sci. 107:3363-3377; Haystead et al.,1989. Nature 337:78-81; Kiguchi et al., 1994. Cell Growth Differ.5:995-1004; Lucocq, 1992. J. Cell Sci. 103:875-880; Ohoka et al.,1993. Biochem. Biophys. Res. Commun. 197:916-921; Suganuma etal., 1988. PNAS 85:1768-1771 Takai et al., 1987. FEBS Lett. 217:81-84

Phenylarsine oxide Han and Kohanski, 1997. Biochem. Biophys. Res. Commun. 239:316-321; Fleming et al., 1996. JBC 271:11009-11015; Krutetskaia et al.,1997. Tsitologiya 39:1116-1130; Kussmann and Przybylski, 1995.Methods Enzymol. 251:430-435

Sodium orthovanadate Brown and Gordon, 1984. JBC 259:9580-9586; Cook et al., 1997.JBC 272:13309-13319; Griffith et al., 1998. JBC 273:10771-10776;Munoz et al., 1992. JBC 267:10381-10388; Seedorf et al., 1995. JBC270:18953-18960; Seglen and Gordon, 1981. JBC 256:7699-7701;Swarup et al., 1982. Biochem. Biophys. Res. Commun. 107:1104-1109

Protease inhibitors


Drug References

continued


A.1K.4


Used inMolecular

Biology Research

Calpain inhibitor I Figueiredo-Pereira et al., 1994. J. Neurochem. 62: 1989-1994; Klafkiet al., 1995. Neurosci Lett. 201:29-32; Milligan et al., 1996. Arch.Biochem. Biophys. 335:388-395

E-64 Banik et al., 1997. Brain Res. 1997 748:205-210; Bush et al., 1997.JBC 272:9086-9092; Sarin et al., 1994. J. Immunol. 153:862-872

Lactacystin Choi et al., 1997. JBC 272:28479-28484; Dick et al., 1996. JBC271:7273-7276; Fenteany et al., 1995. Science 268:726-731; Kim etal., 1997. JBC 272:11006-11010; Oda et al., 1996. Biochem. Biophys.Res. Commun. 219: 800-805

Leupeptin Montenez et al., 1994. Toxicol. Lett. 73:201-208; Sarin et al., 1994. J.Immunol. 153:862-872; Wang et al., 1995. JBC 270:24924-24931

MG-132 Jensen et al., 1995. Cell 83:129-135; Lee and Goldberg, 1996. JBC271:27280-27284; Meerovitch et al., 1997. JBC 272:6706-6713;Salceda and Caro, 1997. JBC 272:22642-22647

PMSF Darby et al., 1998. Biochemistry 37:783-791; Turini et al., 1969. J.Pharmacol. Exp. Ther. 167:98-104; Weaver et al., 1993. Biochem.Cell. Biol. 71:488-500

Pepstatin A Bode and Huber, 1992. Eur. J. Biochem. 204:433-451; Shields et al.,1991. Biochem. Biophys. Res. Commun. 177:1006-1012; Simon et al.,1995. Biochim. Biophys. Acta 1268: 143-151; Yamada et al., 1996. J.Immunol. 157:901-907; Wang et al., 1995. JBC 270:24924-24931

Protein synthesis inhibitorsAnisomycin Dong Chen et al., 1996. JBC 271:6328-6332; Kardalinou et al., 1994.

Mol. Cell. Biol. 14:1066-1074; Sidhu and Omiecinski, 1998. JBC273:4769-4775

Cycloheximide Chow et al., 1995. Exp. Cell Res. 216:149-159; Cotter et al., 1992.Anticancer Res. 12:773-779; Waring, 1990. JBC 265:14476-14480

Emetine Burhans et al., 1991. EMBO J. 10:4351-4360; Gabathuler et al., 1998.J. Cell Biol. 140:17-27; Sidhu and Omiecinski, 1998. JBC 273:4769-4775

Hygromycin Gaken et al., 1992. Biotechniques 13:32-34; Hamada et al., 1994.Curr. Genet. 26:251-255; Lama and Carrasco, 1992. JBC 267:15932-15937

Puromycin Chow et al., 1995. Exp. Cell Res. 216:149-159; Kislauskis et al., 1997.J. Cell Biol. 136:1263-1270; Tachibana et al., 1997. EMBO J. 16:4333-4339; Zhang et al., 1997. Mol. Biol. Cell 8:1943-1954

Transcription inhibitorsActinomycin D Kuhn and Henderson, 1995. JBC 270:20509-20515; McGary et al.,

1997. JBC 272:8628-8634; Wu and Yung, 1994. Eur. J. Pharmacol.270:203-212

α-Amanitin Baumann et al., 1994. Protein Sci. 3:750-756; Rudd and Luse, 1996.\cs52\i JBC 271:21549-21558; Seiser et al., 1995. J. Biol. Chem.270:29400-29406

Other compoundsBrefeldin A Donaldson et al., 1991. J. Cell Biol. 112:579-588; Donaldson et al.,

1992. Nature 360:350-352; Ktistakis et al., 1992. Nature 356:344-346;Lippincott-Schwartz et al., 1990. Cell 60:821-836

Cyclosporin A Su et al., 1997. J. Cell Biol. 139:1533-1543; Sugimoto et al., 1997. J.Biol. Chem. 272:29415-29418; McKeon, 1991. Cell 66:823-826;Nicolli et al., 1996. JBC 271:2185-2192


Drug References

continued


A.1K.5


Desferrioxamine Ben-Shachar et al., 1995. J. Neurochem. 64:718-723; Bergeron et al.,1996. J. Med. Chem. 39:1575-1581; Dang et al., 1994. Res. Commun.Mol. Pathol. Pharmacol. 86: 43-57; Denicola et al., 1995. FreeRadical Biol. Med. 19:11-19; Henderson and Kuhn, 1995. JBC270:20509-20515

2-Deoxyglucose Donaldson et al., 1991. J. Cell Biol. 112:579-588; Hill et al., 1998.JBC 273: 3308-3313; Villalba et al., 1994. JBC 269:2468-2476

Dithiothreitol Braakman et al., 1992. EMBO J. 11:1717-1722; Lodish and Kong,1993. JBC 268:20598-20605; Simons et al., 1995. J. Cell Biol. 130:41-49; Verde et al., 1995. Eur. J. Cell Biol. 67:267-274

Filipin Liu et al., 1997. JBC 272:7211-7222; Schnitzer et al., 1994. J. CellBiol. 127:1217-1232; Silberkang et al., 1983. JBC 258:8503-8511;Smart et al., 1996. J. Cell Biol. 134:1169-1177; Stahl and Mueller,1995. J. Cell Biol. 129:335-344

Fumonisin B1 Merrill et al., 1993. JBC 268:27299-27306; Sandvig et al., 1996. Mol.Biol. Cell. 7:1391-1404; Spiegel and Merrill, 1996. FASEB J. 10:1388-1397; Wang et al., 1991. JBC 266:14486-14490; Wang et al., 1996.PNAS 93:3461-3465

Geneticin Canaani and Berg, 1982. PNAS 79:5166-5170; Southern and Berg,1982. J. Mol. Appli. Genet. 1:327-341; Morris et al., 1996. JBC271:15468-15477.

Leptomycin B Fornerod et al., 1997. Cell 90:1051-1060; Fukuda et al., 1997. Nature390:308-311; Wada et al., 1998. EMBO J. 17:1635-1641; Wolff et al.,1997. Chem. Biol. 4:139-147

Lovastatin Carel et al., 1996. JBC 271:30625-30630; Hancock et al., 1989. Cell57:1167-1177; Jakobisiak et al., 1991. PNAS 88:3628-3632. Mendolaand Backer, 1990. Cell Growth Differ. 1:499-502; Vincent et al., 1991.Biochem. Biophys. Res. Commun. 180:1284-1289

Lysophosphatidic acid An et al., 1998. JBC 273:7906-7910; Jalink et al., 1990. JBC265:12232-12239; Moolenaar, 1995. JBC 270:12949-12952; Zhang et al., 1997. Mol. Biol. Cell. 8:1415-1425

Mastoparan Huber et al., 1997. J. Cell Sci. 110: 2955-2968; Klinker et al., 1996.Biochem. Pharmacol. 51:217-223; Konrad et al., 1995. JBC 270:12869-12876; Schwaninger et al., 1992. J. Cell Biol. 119:1077-1096;Smith et al., 1995. JBC 270:18323-18328

Ouabain Croyle et al., 1997. Eur. J. Biochem. 248:488-495; Peng et al., 1996.JBC 271: 10372-10378; Swann and Steketee, 1989. J. Neurochem.52:1598-1604

PDMP Chen et al., 1995. JBC 270:13291-13297; Inokuchi et al., 1987.Cancer Lett. 38:23-30; Maceyka and Machamer, 1997. J. Cell Biol.139:1411-1418; Rosenwald et al., 1992. Biochemistry 31:3581-3590;Sandvig et al., 1996. Mol. Biol. Cell. 7:1391-1404; Uemura et al.,1990. J. Biochem. 108:525-530

Pertussis toxin An et al., 1998. JBC 273:7906-7910; Hewlett et al., 1983. Infect.Immun. 40:1198-1203; Jakobs et al., 1984. Eur. J. Biochem. 140:177-181; Kopf and Woolkalis, 1991. Methods Enzymol. 195:257-266;Luttrell et al., 1997. JBC 272:31648-31656

Phorbol esters Janecki et al., 1998. JBC 273:8790-8798; Macfarlane and O’Donnell,1993. Leukemia 7:1846-1851; Niedel et al., 1983. PNAS 80:36-40;Nishizuka, 1986. Science 233:305-312; Oishi and Yamaguchi, 1994. J.Cell. Biochem. 55: 168-172; Schmidt et al., 1998. JBC 273:7413-7422; Tepper et al., 1995. PNAS 92:8443-8447


Drug References

continued


A.1K.6


Used inMolecular

Biology Research

DRUGS COMMONLY USED IN MOLECULAR BIOLOGY

A23187Calcium ionophore; forms stable complexes with divalent cations and increases theirpassage across biological membranes. Useful tool for increasing intracellular cal-cium concentration. The effectiveness of A23187 is dependent on the presence ofextracellular calcium. Can be used as a fluorescent probe for investigating proteinhydrophobicity. 4-Bromo-A23187 is a nonfluorescent derivative.Soluble in: DMSO, methanolStock concentration: 100 mM (store at 4°C protected from light)Working concentration: 0.1 to 20 µMDuration of incubation: 2 min to 24 hr

Aggregates over time in aqueous systems.

Actinomycin DInhibits transcription by complexing with deoxyguanosine residues on DNA andblocking the movement of RNA polymerase. A potent inducer of apoptosis in manycell lines. However, actinomycin D has also been shown to suppress programmedcell death of PC12 cells induced by the topoisomerase II inhibitor etoposide.Soluble in: MethanolStock concentration: 100 mM (store at 4°C)Working concentration: 1 to 5 µMDuration of incubation: 5 min to 24 hr

Rapamycin Brown et al., 1994. Nature 369:756-758Jefferies et al., 1997. EMBO J. 16:3693-3704Kozlovsky et al., 1997. J. Biol. Chem. 272:33367-33372Liu et al., 1991. Cell 66:807-815Sabers et al., 1995. J. Biol. Chem. 270:815-822

Sodium azide Bhat et al., 1996. JBC 271:32551-32556; Donaldson et al., 1991. J.Cell Biol. 112:579-588; van Klompenburg et al., 1997. EMBO J.16:4261-4266

Sodium butyrate Calabresse et al., 1993. Biochem. Biophys. Res. Commun. 195:31-38;Gonzalez-Garay and Cabral, 1996. J. Cell Biol. 135:1525-1534; Russoet al., 1997. Biochem. Biophys. Res. Commun. 233:673-677; Vaziri etal., 1996. JBC 271:25921-25927; White et al., 1995. J. Cell Sci.108:441-455

Trifluoperazine Aussel et al., 1995. JBC 270:8032-8036; Massom et al., 1990.Biochemistry 29:671-681; Rao, 1987. Biochem. Biophys. Res.Commun. 148:768-775

Valinomycin Inai et al., 1997. Cell Struct. Funct. 22:555-563; Loiseau et al., 1997.Biochim. Biophys. Acta. 1330:39-49; Orlov et al., 1994. FEBS Lett.345:104-106; Szabo et al., 1997. JBC 272:23165-23171

W-7 de Figueiredo and Brown, 1995. Mol. Biol. Cell. 6:871-887; Hidaka etal., 1981. PNAS 78:4354-4357; Hunziker, 1994. JBC 269:29003-29009; Wolf and Gross, 1996. JBC 271:20989-20992


Drug References


A.1K.7


α-AmanitinActs as a potent and specific inhibitor of mRNA synthesis by binding preferentiallyto RNA polymerase II. At high concentrations also inhibits RNA polymerase III.Soluble in: Methanol, waterStock concentration: 2 to 10 mg/ml (store at 4°C protected from light)Working concentration: 1 to 10 µg/ml (pol II) to 200 µg/ml (pol III)Duration of incubation: 15 to 60 min

Ammonium chloride (NH4Cl)Permeant weak base. Used to neutralize acidic endomembrane compartments.Inhibits synthesis of sphingoid bases.Soluble in: Water (freely soluble)Stock concentration: 5 M (store at 4°C)Working concentration: 1 to 50 mMDuration of incubation: Effective within 15 sec

AnisomycinInhibits protein synthesis by blocking the peptidyl transferase step during transla-tion. Activates p54 (JNK2) and MAP kinases. May be a useful tool to studycytoplasmic signals that result in nuclear signaling and c-fos and c-jun induction.Also known to induce apoptosis in U937 cells.Soluble in: DMSOStock concentration: 100 µg/ml (store at 4°C)Working concentration: 50 ng/ml to 1 µg/ml.Duration of incubation: 30 min to 16 hr, depending on properties studied

AphidicolinCell synchronization reagent. Reversible inhibitor of DNA polymerase α and δ;blocks cell cycle at early S phase. Potentiates apoptosis induced by arabinosylnucleosides in leukemia cell lines.Soluble in: DMSO, methanolStock concentration: 2 mg/ml (store at 4°C)Working concentration: 0.5 to 100 µg/ml.Duration of incubation: 12 to 24 hr

Ara-C (cytosine arabinoside)Inhibits DNA synthesis. S-phase-toxic reagent whose active metabolite (ara-CTP)is a substrate for DNA polymerases and is incorporated into DNA. Anticancer,antiviral agent that is especially effective against leukemias. Induces apoptosis inhuman myeloid leukemia cells and in rat sympathetic neurons.Soluble in: WaterStock concentration: 20 mg/ml (store at 4°C)Working concentration: 0.1 to 1 µg/mlDuration of incubation: >3 hr

Bafilomycin A1

A potent and specific inhibitor of vacuolar-type H+-ATPases. Valuable tool fordistinguishing different types of ATPases. Blocks lysosomal trafficking in macro-phages.Soluble in: DMSOStock concentration: 50 µM (store at −20°C protected from light)Working concentration: 10 to 100 nMDuration of incubation: 10 min to 2 hr


A.1K.8


Used inMolecular

Biology Research

BAPTACa2+ chelator with a 105-fold greater affinity for Ca2+ than for Mg2+; can be used tocontrol the level of both intracellular (using its membrane-permeant AM ester) andextracellular Ca2+.Soluble in: DMSOStock concentration: 1 to 10 mM (store at −20°C in aliquots, protected from

light; avoid repeated freeze-thawing)Working concentration: (BAPTA-AM): 1 to 20 µMDuration of incubation: 15 to 60 min at 20° to 37°C

Before incubation with BAPTA, wash cells 2 to 3 times with serum-free medium (serum maycontain esterase activity). The cell-loading medium should also be free of amino acids or bufferscontaining primary or secondary amines that may cleave the AM esters and prevent loading.

Bisindolylmaleimide I (GF 109203X)A highly selective cell-permeant protein kinase C (PKC) inhibitor that is structurallysimilar to staurosporine, but has higher selectivity. May inhibit protein kinase A athigh concentrations. Acts as a competitive inhibitor for the ATP-binding site of thePKC catalytic domain. Since ATP levels are generally very high in cells, the potencyof bisindolylmaleimide I is reduced accordingly in whole-cell assays.Soluble in: DMSOStock concentration: 2 mM (store at ≤4°C)Working concentration: 20 nM to 1 µMDuration of incubation: 15 min to 6 hr

Water-soluble salts are available.

Brefeldin AInhibits GTP nucleotide exchange onto several members of the ARF (ADP ribosy-lation factor) family. Inhibits binding of the cytosolic coatomer (COPI) complex toGolgi membranes; induces the rapid redistribution of the Golgi apparatus into theER; blocks transport out of the ER in a number of cell lines. Reversible.Soluble in: MethanolStock concentration: 1 to 20 mM (store at −20°C)Working concentration: 1 to 5 µMDuration of incubation: 5 min to 24 hr; effects are rapid (30 sec)

8-Bromo–cyclic AMPCell-permeant cyclic AMP analog. Activates protein kinase A. Increased resistanceto degradation by cellular phosphodiesterases as compared to cyclic AMP.Soluble in: WaterStock concentration: 100 mM (store at −20°C)Working concentration: 10 to 500 µMDuration of incubation: Up to 24 hr

Calpain inhibitor I (ALLN)Inhibitor of calpain I, calpain II, cathepsin B, and cathepsin L. A peptide aldehyde,which inhibits neutral cysteine proteases and the proteosome. Protects againstneuronal damage caused by hypoxia and ischemia. Inhibits proteolysis of IkB bythe ubiquitin-proteosome complex. Inhibits cell-cycle progression at G1/S andmetaphase/anaphase in CHO cells by inhibiting cyclin B degradation. Membrane-permeant due to low molecular weight and lack of charged residues.Soluble in: DMSO, methanol, dimethylformamideStock concentration: 25 mM (store at 4°C)Working concentration: 25 to 100 µMDuration of incubation: 1 to 18 hr


A.1K.9


Calphostin CPotent and highly selective inhibitor of protein kinase C (PKC; Ki = 50 nM).Competes with phorbol esters and diacylglycerol for binding to the PKC regulatorydomain. Does not compete with Ca2+ or phospholipids. At higher concentrationsinhibits myosin light chain kinase (Ki > 5 µM), protein kinase A (Ki > 50 µM), proteinkinase G (Ki > 25 µM), and p60v-src (Ki > 50 µM).Soluble in: DMSOStock concentration: 1 mM (store 4°C protected from light)Working concentration: 10 nM to 3 µMDuration of incubation: 15 to 60 min

Brief exposure to visible light in the presence of PKC is required for PKC inhibition bycalphostin C. See Table 1 (Selectivity of Selected Kinase Inhibitors) in the Biomol Catalogand Handbook, 5th ed., and Technical Note #11 from Alexis Biochemicals for additionalinformation.

Calyculin APotent cell-permeant inhibitor with high specificity for the Ser/Thr protein phos-phatases 1 and 2A. Calyculin A is 20 to 300 times more potent than okadaic acid asa PP-1 class phosphatase inhibitor. Stimulates contraction of smooth muscle,induces intracellular protein phosphorylation in cultured human keratinocytes, andinhibits apoptosis.Soluble in: DMSO, ethanolStock concentration: 10 µM (store at −20°C protected from light and moisture)Working concentration: 0.5 to 50 nMDuration of incubation: 15 min to 2 hr

See Technical Note #19 from Alexis Biochemicals for additional information.May cause cellrounding.

CamptothecinA reversible DNA topoisomerase I inhibitor. Induces breaks at replication forks bybinding to and stabilizing the topoisomerase-DNA covalent complex. Causes S-phase cytotoxicity. Posesses anti-leukemic and anti-tumor properties. Inhibits tat-mediated transactivation of HIV-1.Soluble in: DMSOStock concentration: 1 to 10 mM (store at 4°C)Working concentration: 0.1 to 10 µMDuration of incubation: Literature shows use from 15 min to 24 hr

CastanospermineInhibitor of α- and β-glucosidases; inhibitor of glycoprotein processing. Preventscalnexin and calreticulin binding to N-linked glycans on newly synthesized glyco-proteins. Inhibits HIV infectivity.Soluble in: WaterStock concentration: 1 mM (store at 4°C)Working concentration: 1 to 5 µMDuration of incubation: 15 min to 3 hr


A.1K.10


Used inMolecular

Biology Research

CCCP (carbonyl cyanide-m-chlorophenyl hydrazone)Proton ionophore. Uncoupling agent for oxidative phosphorlyation that inhibitsmitochondrial function. Approximately 100 times as effective as 2,4-dinitrophenolat collapsing membrane potential. Inhibits transport processes and depressesgrowth.Soluble in: DMSO, ethanolStock concentration: 1 to 10 mM (store at 4°C)Working concentration: 1 to 5 µMDuration of incubation: 5 to 15 min

Chelerythrine chloridePotent, selective, cell-permeant inhibitor of protein kinase C (Ki = 0.66 µM). Actson the catalytic domain. Chelerythrine shows competitive kinetics with PKC sub-strates, but is not competitive with ATP. Thus, the high concentration of ATP withincells should not lower the potency of chelerytherine in whole cells as compared withthat seen in purified enzyme preparations. Inhibits thromboxane formation andphosphoinositide metabolism in platelets. Induces apoptosis in HL-60 cells.Soluble in: DMSOStock concentration: 10 mM (store at −20°C)Working concentration: 1 µMDuration of incubation: 15 min to 2 hr

See Technical Note #8 from Alexis Biochemicals for additional information.

ChloroquineTertiary amine that accumulates within and neutralizes the pH of acidic organelles;various effects on phagosome-endosome and phagosome-lysosome fusion. Anti-malarial drug that works via carrier-mediated uptake in P. falciparum. May activateprotein kinases.Soluble in: WaterStock concentration: 1 to 10 mg/ml (store at room temperature)Working concentration: 10 to 200 µg/mlDuration of incubation: 15 min to 2 hr

Cholera toxinContains a single A subunit (mol. wt. = 29 kDa) and a B subunit (mol. wt. = 55 kDa)containing five B polypeptide chains. The B subunit binds to GM1 gangliosidereceptors on the surface of cells and facilitates transport of the A subunit throughthe membrane. The A subunit catalyzes the ADP-ribosylation of an arginine residueon the α subunit of heterotrimeric G proteins (primarily Gs), reducing its intrinsicGTPase activity. Toxicity results from activation of membrane-bound adenylatecyclase. Consequently, increased intracellular cAMP levels result in increasedelectrolyte transport out of the cell and water loss. Cholera toxin requires ADP-ri-bosylation factor (ARF) for maximal activity.Soluble in: WaterStock concentration: 1 mg/ml (store at 4°C, do not freeze)Working concentration: 100 ng/ml to 2 µg/mlDuration of incubation: 2 to 24 hr


A.1K.11


ColcemidCell synchronization agent. Depolymerizes microtubules and limits microtubuleformation. Low concentrations inactivate spindle dynamics. Induces apoptosis byblocking mitosis in HeLa S3 cells. Colcemid is a less toxic derivative of colchicine.Soluble in: EthanolStock concentration: 1 mM (store at or below room temperature)Working concentration: 1 to 10 µMDuration of incubation: 1 to 24 hr, depending on process studied

ColchicineInhibitor of mitosis, used in cell-division studies. Disrupts microtubules and inhibitstubulin polymerization. Induces apoptosis in PC 12 cells and in cerebellar granule cells.Soluble in: EthanolStock concentration: 1 mM (store at or below room temperature protected from

light and moisture)Working concentration: 1 to 10 µMDuration of incubation: 1 to 24 hr, depending on process studied

Concanamycin BHighly specific and sensitive inhibitor of vacuolar-type H+-ATPases (Ki = 20 pM).Related to concanamycin A (folimycin). More potent and specific than bafilomycinA1. Inhibits acidification of organelles such as lysosomes and the Golgi apparatus.Blocks cell-surface expression of viral glycoproteins without affecting their synthesis.Soluble in: Methanol, ethanolStock concentration: 10 µM (store at −20°C protected from light)Working concentration: 50 nMDuration of incubation: 5 min to 1 hr

CycloheximideInhibits protein synthesis in eukaryotes but not prokaryotes. Blocks the translocationstep during translation. Induces apoptosis in a number of cell types. However, itinhibits DNA cleavage in rat thymocytes treated with thapsigargin and ionomycin.Soluble in: Water, ethanol, methanolStock concentration: 10 mg/ml (store at or below room temperature)Working concentration: 1 to 100 µg/ml, depending on cell typeDuration of incubation: Effective within 10 min

To achieve >90% inhibition of protein synthesis, only 1 to 10 g/ml is required in CHO andHeLa cells, but 100 g/ml is required in COS cells.

Cyclosporin A (CsA)Cyclic oligopeptide with immunosuppressant properties. Induces apoptosis in somecell types, while inhibiting apoptosis in others. A complex of cyclosporin A andcyclophilin inhibits protein phosphatase 2B (calcineurin) with affinity at the nano-molar level. Inhibits nitric oxide synthesis induced by interleukin-1α, lipopolysac-charides, and TNFα.Soluble in: Ethanol, methanolStock concentration: 1 to 5 mM (store at 4°C)Working concentration: 0.1 to 10 µMDuration of incubation: Used anywhere from 15 min to 24 hr

Cytochalasin BCell-permeant fungal toxin that blocks the formation of contractile microfilaments.Shortens actin filaments by blocking monomer addition at the barbed (fast-growing)

continued


A.1K.12


Used inMolecular

Biology Research

end of polymers. Inhibits cytoplasmic division, cell movement, phagocytosis,platelet aggregation, and glucose transport.Soluble in: DMSO, ethanolStock concentration: 10 mM (store at −20°C protected from light)Working concentration: 1 to 20 µMDuration of incubation: 15 min to 2 hr

Cytochalasin DApproximately 10-fold more potent than cytochalasin B in inhibiting actin filamentfunction. Does not inhibit sugar transport in cells. Modulates CD4 cross-linking inT lymphocytes and increases intracellular Ca2+. Exhibits antitumor activity.Soluble in: DMSOStock concentration: 10 mM (store at −20°C protected from light)Working concentration: 1 to 20 µMDuration of incubation: 15 min to 2 hr.

Desferrioxamine (DFO)Iron-chelating agent. Commonly used in therapy as a chelator of ferric iron in ironoverload disorders. Protects against dopamine-induced cell death. Also interfereswith hydroxy-radical formation. Shows an antiproliferative effect on vascularsmooth muscle cells.Soluble in: DMSO; slightly soluble in waterStock concentration: 10 to 50 mM (store at 4°C)Working concentration: 10 µM to 2 mMDuration of incubation: Up to 18 hr

2-DeoxyglucoseNonmetabolizable derivative of glucose. Competes with glucose for the GLUT-2transporter; phosphorylation of 2-deoxyglucose by hexokinase effectively inhibitsglucose flux through the glycolytic pathway. Used in combination with sodium azideor oligomycin to reduce cellular ATP levels. Blocks inhibition of IL-1 release byhigh glucose levels in RAW 264.7 cells.Soluble in: WaterStock concentration: 1 M (store at 4°C)Working concentration: 5 to 50 mMDuration of incubation: 15 min to 3 hr

DeoxymannojirimycinCompetitive α-mannosidase I inhibitor that blocks conversion of high mannoseforms to complex oligosaccharides. Inhibits mammalian Golgi α-mannosidase I (anα-1,2-mannosidase). Other rat liver mannosidases are not significantly affected (α-1,2–specific ER mannosidase is only inhibited 2% to 5% by 100 mM deoxyman-nojirimycin, and Golgi α-mannosidase II is inhibited ∼14%).Soluble in: WaterStock concentration: 100 mM (store at −20°C)Working concentration: 1 to 5 mMDuration of incubation: Anywhere from 30 min to 24 hr

DeoxynojirimycinSpecific glucosidase inhibitor. Inhibits endoplasmic reticulum trimming glu-cosidases I and II, which sequentially remove three glucose residues fromGlc3Man9GlcNAc2 in N-linked glycan biosynthesis. Prevents calnexin and cal-reticulin binding to N-linked glycoproteins within the ER.

continued


A.1K.13


Soluble in: WaterStock concentration: 100 mM (store at 4°C)Working conditions: 1 to 5 mM.Duration of incubation: 15 min to 24 hr

At concentrations >1 mM, deoxynojirimycin may inhibit lipid-linked oligosaccharide bio-synthesis as well as trimming. In such cases, N-methyldeoxynojirimycin may be a moreeffective inhibitor, possibly owing to an increased ability to cross cell membranes, affordedby the N-methyl group.

Dibutyryl cyclic AMPHighly membrane-permeant cAMP analog resistant to phosphodiesterase cleavage.Constitutive activator of protein kinase A. This product releases butyrate due tointracellular and extracellular esterase action. Butyrate may have its own distinctbiological effects (see sodium butyrate).Soluble in: DMSO, ethanolStock concentration: 1 M (store at −20°C)Working concentration: 100 µM to 1 mMDuration of incubation: Anywhere from 1 to 48 hr

Dithiothreitol (DTT; Cleland’s reagent)Cell-permeant protective agent for SH groups; maintains monothiols completely inthe reduced state and reduces disulfides quantitatively. DTT interferes with thefolding and export of proteins located in the endoplasmic reticulum, but it does notprevent the transfer from the intermediate compartment to the Golgi complex.Reversible.Soluble in: Water, ethanolStock concentration: 1 M (store at 4°C)Working concentration: 1 to 10 mMDuration of incubation: 1 min to several hours

E-64Irreversible inhibitor of cysteine proteases (papain and cathepsins B and L). Has noaction on cysteine residues in other proteins.Soluble in: WaterStock concentration: 1 mg/ml (store at −20°C)Working concentration: 0.5 to 10 µg/mlDuration of incubation: Up to 24 hr

EmetineIrreversibly blocks protein synthesis by inhibiting movement of ribosomes alongmRNA. Stimulates rapid and differential phosphorylation of the stress-activatedprotein kinase/c-Jun kinase (SAPK/JNK) pathway. Prevents apoptosis in several celllines. In primary rat hepatocytes, the relative potency of inhibition of several proteinsynthesis inhibitors is in the order: emetine > anisomycin > cycloheximide >puromycin, with puromycin exhibiting only marginal inhibition at a concentrationof 1 µM. In fact, 90% to 95% inhibition of protein synthesis was achieved only withemetine and anisomycin, at 10 µM concentrations. Cycloheximide and puromycinexerted only 80% and 60% inhibition, respectively, at a similar concentration.Soluble in: EthanolStock concentration: 10 to 100 mM (store at 4°C protected from light)Working concentration: 10 to 20 µMDuration of incubation: 15 to 30 min; can incubate 24 hr


A.1K.14


Used inMolecular

Biology Research

Etoposide (VP-16)Topoisomerase II inhibitor. Stabilizes the covalent complexes of topoisomerase IIwith DNA. Has major activity against a number of tumors, including germ cellneoplasms, small cell lung cancer, and malignant lymphoma. Induces apoptosis inmouse thymocytes and HL-60 cells. Activates PKCα.Soluble in: DMSOStock concentration: 100 to 500 mM (store at room temperature)Working concentration: 50 to 200 µMDuration of incubation: 1 to 24 hr

FilipinA cholesterol-binding fluorochrome. Specific for unesterified cholesterol. Bindsand removes cholesterol from cell surface membranes. Reversibly disassemblescaveolae.Soluble in: MethanolStock concentration: 500 µg/ml (store 4°C protected from light)Working concentration: 5 to 50 µg/mlDuration of incubation: 1 hr

ForskolinActivates adenylate cyclase by interacting directly with the catalytic subunit. Leadsto an increase in the intracellular concentration of cAMP. Several forskolin deriva-tives are available having different and improved properties. Enhances detoxifica-tion of brefeldin A.Soluble in: DMSO, ethanolStock concentration: 10 to 100 mM (store at 4°C)Working concentration: 10 µM (to increase cAMP levels); 100 µM (to inhibit bre-

feldin A)Duration of incubation: Depending on assay, incubate cells 15 min to 12 hr

Fumonisin B1

Inhibits sphingolipid biosynthesis via inhibition of sphingosine N-acyltransferase(ceramide synthase). Sphingomyelin biosynthesis is preferentially inhibited versusglycosphingolipids in neuronal cells. Inhibits the butyric acid–induced increase intransport of cell-associated Shiga toxin to the Golgi apparatus and the ER. Inducesapoptosis in monkey kidney cells.Soluble in: methanolStock concentration: 10 to 100 mM (store at 4°C)Working concentration: Anywhere from 1 to 100 µMDuration of incubation: 15 min to 18 hr, depending on process studied

Geneticin (G418)Aminoglycoside toxic to bacteria, yeast, higher plants, protozoa, and mammalian cells.Used for the selection and maintenance of eukaryotic cells stably transfected withthe neomycin (neo) resistance genes from transposons Tn5 and Tn601.Soluble in: Water or culture mediumStock concentration: 2 mg/ml (active G418) in cell culture medium, adjust pH to

∼7.4 (store at 4°C)Working concentration: Usually 50 to 1000 µg/ml (optimal concentration must

be determined experimentally and varies with the cell type used)Duration of incubation: >1 week

In cell types with relatively stable genomes (e.g., CHO), continuous incubation in geneticinis not generally necessary once stable cells have been selected. Expression in stable cellswith down-regulated viral promoters can be enhanced with sodium butyrate.


A.1K.15


GenisteinInhibits protein tyrosine kinases by acting as a competitive inhibitor of ATP. PreventsEGF-stimulated tyrosine phosphorylation in A431 cells, as well as inhibiting kinasesin other cultured cells.Soluble in: DMSOStock concentration: 100 to 500 mM (store at −20°C)Working concentration: 50 to 300 µMDuration of incubation: 15 min to 1 hr

See Table II (Selectivity of Tyrosine Protein Kinase Inhibitors) in the Biomol Catalog andHandbook, 5th ed.

H-7A broad-based, cell-permeant serine/threonine kinase inhibitor. Inhibits protein kinaseC (Ki = 6.0 µM), protein kinase A (Ki = 3.0 µM), protein kinase G (Ki = 5.8 µM), andmyosin light chain kinase (Ki = 97 µM). Induces apoptotic DNA fragmentation andcell death in HL-60 cells. Numerous analogs with different selectivities are available.Soluble in: Water Stock concentration: 100 mM (store at 4°C)Working concentration: 10 to 100 µMDuration of incubation: 15 min to 3 hr

See Table I (Selectivity of Selected Kinase Inhibitors) in the Biomol Catalog and Handbook,5th ed., and Technical Note #10 from Alexis Biochemicals.

Herbimycin AAn irreversible and selective cell-permeant protein tyrosine kinase inhibitor; reactswith thiol groups. It is effective on Src, Yes, Fps, Ros, Abl, and ErbB oncogene products.Inhibits PDGF-induced phospholipase D activation in a dose-dependent manner.Soluble in: DMSOStock concentration: 10 mM (store at −20°C protected from light)Working concentration: 1 to 10 µMDuration of incubation: 15 min to 1 hr

HydroxyureaAntineoplastic reagent. Blocks DNA synthesis by inhibiting ribonucleotide reduc-tase; accumulates cells at G1/S interface.Soluble in: WaterStock concentration: 1 M (store at 4°C)Working concentration: 50 µM to 1 mMDuration of incubation: Up to 24 hr

HygromycinInhibitor of both prokaryotic and eukaryotic protein synthesis; inhibits at thetranslocation step on 70S ribosomes and causes misreading of mRNA. The E. colihph hygromycin B–resistant gene is widely used for selection of recombinant clonesin a variety of cell types.

Hygromycin B is sold as an aqueous solution. Actual activity and concentration are givenfor each lot of product. Concentrations used must be experimentally determined.

IonomycinCa2+ ionophore. Useful for increasing intracellular Ca2+ concentrations and inmeasurement of cytoplasmic free Ca2+. More effective than A23187 and is nonfluo-rescent.Soluble in: DMSO and methanolStock concentration: 1 mM (store at 4°C protected from light)

continued


A.1K.16


Used inMolecular

Biology Research

Working concentration: Usually 1 µMDuration of incubation: 15 to 30 min.

KN-62Potent and selective inhibitor of Ca2+/calmodulin kinase II (Ki = 0.9 µM), displayinga Ki for CaM kinase II more than 2 orders of magnitude lower than those for proteinkinase C, protein kinase A, and myosin light chain kinase. A second inhibitor ofCaM kinase II, KN-93, is more soluble in water and equally selective for CaM kinaseII (Ki = 0.3 µM). Prevents agonist-mediated activation of Ins(1,4,5)P3 3-kinase.Inhibits differentiation of 3T3-L1 embryonic fibroblasts to adipocytes. Inactiveanalogs are available.Soluble in: DMSOStock concentration: 10 mM (store at 4°C)Working concentration: 2 to 10 µMDuration of incubation: 30 min (can incubate cells up to 48 hr)

LactacystinA cell-permeant and irreversible proteosome inhibitor. Blocks proteosome activityby targeting the catalytic β subunit. Induces neurite outgrowth in Neuro 2A mouseneuroblastoma cells and inhibits progression of synchronized Neuro 2A cells andMG-63 human osteosarcoma cells beyond the G1 phase of the cell cycle. InhibitsNFκB activation. Has revealed the role of the proteosome in the degradation of manyER proteins.Soluble in: DMSOStock concentration: 10 mM (store at −20°C)Working concentration: 10 to 20 µMDuration of incubation: 1 to 12 hr

Latrunculin AInhibits actin polymerization and disrupts microfilament organization as well asmicrofilament-mediated processes; 10 to 100-fold more potent than cytochalasins.Whereas cytochalasins induce dissolution of F-actin and stress-fiber contraction infibroblasts in culture, the latrunculins (A and the less potent B) cause a shorteningand thickening of stress fibers. In addition, the latrunculins sequester actin mono-mers, whereas with the cytochalasins, actin remains in an oligomer form. Thus, thetwo classes of compounds may have different target sites. Reversible.Soluble in: DMSO, ethanolStock concentration: 10 mg/ml (store at −20°C)Working concentration: 0.2 to 10 µg/mlDuration of incubation: 1 to 12 hr

Leptomycin BA potent and specific inhibitor of the NES-dependent nuclear export of proteins;binds to the export receptor CRM1. Exhibits antifungal and antitumor effects,inhibits the nucleo-cytoplasmic translocation of the human immunodeficiency virustype 1 regulatory protein Rev, and exhibits significant antiproliferative activity.Soluble in: EthanolStock concentration: 100 µM to 1 mM (store at −20°C)Working concentration: 10 to 100 nMDuration of incubation: 30 min to 3 hr


A.1K.17


LeupeptinA reversible inhibitor of trypsin-like and cysteine proteases (including trypsin,plasmin, proteinase K, papain, thrombin, and cathepsin A and B). Inhibits activa-tion-induced programmed cell death in T lymphocytes.Soluble in: WaterStock concentration: 1 to 10 mM (store at −20°C)Working concentration: 10 to 100 µMDuration of incubation: 15 min to several hours

LovastatinAn antihypercholesterolemic agent and inhibitor of 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase; depletes endogenous pools of mevalonic acid,thereby blocking protein isoprenylation and cholesterol synthesis. Has a number ofcellular effects. Blocks N-ras oncogene–induced neuronal differentiation, inhibitsgrowth factor signaling and causes cells to arrest in late G1 phase.Soluble in: DMSO, ethanolStock concentration: 4 mg/ml (store at −20°C)Working concentration: 20 µMDuration of incubation: 6 to 24 hr

Lysophosphatidic acid (LPA)Activates a number of signaling pathways and processes via heterotrimeric Gproteins (primarily Gi and Gq) including: inhibition of adenylate cyclase, activationof Ras and the Raf/MAP kinase pathway, stimulation of phospholipases C and D,and stress-fiber formation through the activation of Rho.Solubility: A stock solution may be prepared at 10 mg/ml in 95:5:5 chloro-form/methanol/acetic acid (gives a clear solution). Solubility in dimethysulfoxide(DMSO) or ethanol is limited. The sodium salt of oleoyl-LPA is reported to bereadily soluble at 5 mg/ml (∼11 mM) in calcium and magnesium-free buffers (Jalinket al., 1990). Solubilization has also been achieved (Seufferlein and Rozengurt,1994) in phosphate-buffered saline (PBS), pH 7.4, or calcium- and magnesium-freeDulbecco’s PBS (CMF-DPBS), pH 7.4 (see APPENDIX 2 for recipes), at up to 3 mM(0.14 mg/ml) in the presence of 0.1% (w/v) BSA (essentially fatty-acid free).Storage. LPA should be stable in solution under neutral conditions. Freezer storage isrecommended for solutions or aqueous preparations. Maintaining the product underan inert atmosphere (nitrogen or argon) may be appropriate for some applications.Working concentration. Anywhere from 500 nM (1-hr incubation) to 100 µM(15-min incubation).

LY294002 (PI 3-kinase inhibitor)Reversible inhibitor of phosphatidylinositol-3-kinase that acts on the ATP-bindingsite of the enzyme. Does not affect the activity of EGF receptor kinase, MAP kinase,PKC, PI4-kinase, S6 kinase, and c-src. Blocks proliferation of cultured rabbit aorticsmooth muscle cells without inducing apoptosis. Wortmannin is more selective andmore potent, but is irreversible.Soluble in: DMSO and ethanolStock concentration: 1 to 10 mM (store in aliquots at −20°C)Working concentration: 1 to 2 µMDuration of incubation: 15 min to 3 hr


A.1K.18


Used inMolecular

Biology Research

MastoparanRelatively cell-permeant synthetic peptide capable of directly activating pertussistoxin–sensitive G proteins by a mechanism analogous to that of G-protein-coupledreceptors. Acts preferentially on Gi and Go rather than Gs. Stimulates insulinsecretion in permeabilized cells, and can increase intracellular Ca2+ levels. Inhibitscalmodulin and activates phospholipase A2.Soluble in: WaterStock concentration: 1 mM (store at −20°C)Working concentration: 10 to 50 µMDuration of incubation: 15 min to 1 hr

Microcystin-LRCyclic heptapeptide; potent inhibitor of protein phosphatases 1 and 2A (PP-1 andPP-2A). Unlike okadaic acid, microcystin-LR is equally effective on both PP-1 (Ki

= 1.7 nM) and PP-2A (Ki = 0.04 nM). Has no effect on protein kinases, making ituseful for reducing the effect of contaminating phosphatases in protein kinaseassays. It is not cell-permeant, but can enter hepatocytes via the multispecificorganic anion transporter.Soluble in: DMSO, ethanol, methanolStock concentration: 1 mM (store at −20°C)Working concentration: 1 to 5 µM in hepatic cells; 10 µM in vitroDuration of incubation: 15 min to 1 hr

MG-132A potent, reversible and cell-permeant proteosome inhibitor. Reduces the degrada-tion of ubiquitin-conjugated proteins by the 26S complex without affecting itsATPase or isopeptidase activities. Has been used to implicate the proteosome in thebreakdown of membrane proteins, including the CFTR, within the ER (see alsolactacystin). Inhibits NFκB activation.Soluble in: DMSOStock concentration: 10 to 100 mM (store at −20°C)Working concentration: 20 to 200 µMDuration of incubation: 30 min to 24 hr

ML-7 (MLCK inhibitor)Potent, cell- permeant, and selective inhibitor of myosin light chain kinase (Ki = 300nM). Inhibits protein kinase A (Ki = 21 µM) and protein kinase C (Ki = 42 µM) atmuch higher concentrations.Soluble in: DMSO, ethanol, waterStock concentration: 100 to 500 mM (store at 4°C protected from light)Working concentration: 10 to 50 µMDuration of incubation: 15 min to 1 hr

See Table I (Selectivity of Selected Kinase Inhibitors) in the Biomol Catalog and Handbook,5th ed.

L-MimosineAn inhibitor of DNA replication that may act by preventing the formation ofreplication forks. L-mimosine blocks the camptothecin-induced apoptosis of PC12cells, whereas aphidicolin does not.Soluble in: WaterStock concentration: 10 mM (store at room temperature)Working concentration: 25 to 400 µMDuration of incubation: 2 to 24 hr


A.1K.19


MonensinPolyether antibiotic that functions as an Na+ ionophore. Forms stable complexeswith monovalent cations that are able to cross cell membranes. Inhibits glycoproteinsecretion by blocking transport through the Golgi. Neutralizes acidic endomem-brane compartments. Reduces sphingomyelinase activity.Soluble in: DMSO, methanolStock concentration: 2 to 30 mM (store at 4°C)Working concentration: 1 to 30 µMDuration of incubation: Effective within seconds; use up to 3 hr.

NigericinDual antiporter ionophore that acts as a K+/H+ exchanger. Stimulates Ca2+ releasefrom mitochondrial stores by disruption of membrane potential. Allows adjustmentof cytoplasmic pH (when combined with K+ ionophore such as valinomycin).Soluble in: EthanolStock concentration: 1 mg/ml (store at 4°C)Working concentration: 1 to 10 µMDuration of incubation: Effective within 2 to 5 min

NocodazoleHas specific antimicrotubular activity for mammalian cells in culture. Promotesmicrotubule depolymerization. Nanomolar concentrations alter microtubule dy-namics and interfere with fibroblast locomotion without affecting polymer levels.Arrests cells in mitosis.Soluble in: DMSOStock concentration: 10 to 30 mM (store at room temperature)Working concentration: 50 nM (low concentrations) to 30 µM (for effective mi-

crotubule depolymerization)Duration of incubation: For rapid depolymerization of microtubules, preincubate

cells on ice with nocodazole for 15 min; use up to 24 hr.

Okadaic acidPotent inhibitor of protein phosphatases, especially the PP-1 class (Ki = 10-15 nM)and PP-2A class (Ki = 0.1 nM), in numerous cell types. Does not affect the activityof acid or alkaline tyrosine phosphatases. It mimics the effects of insulin, enhancesneurotransmitter release, causes vasodilation, and is a potent tumor promoter.Induces dispersal of the Golgi apparatus. Okadaic acid is a useful tool for studyingcellular processes regulated by serine/threonine phosphorylation.Soluble in: DMSO, ethanol, methanolStock concentration: 1 mM (store at −20°C protected from light)Working concentration: 50 to 200 nMDuration of incubation: 15 min to 2 hr

May cause cell rounding. See Technical Note #18 from Alexis Biochemicals for additionalinformation.

OlomoucineAdenine derivative that acts as a competitive inhibitor for ATP binding and inhibitsp34cdc2/cyclin B (Ki = 7 µM) as well as several other CDKs at low concentrations.Does not significantly affect the activity of other protein kinases at 1 mM. InhibitsDNA synthesis in IL-2 stimulated T lymphocytes. Also used to synchronize cells inG1. Can affect microtubule dynamics at higher concentrations.Soluble in: DMSOStock concentration: 100 mM (store in aliquots at −20°C)Working concentration: Usually 10 µM (up to 100 µM) continued


A.1K.20


Used inMolecular

Biology Research

Duration of incubation: 15 min to 24 hr, depending on process studied.


OuabainSelective Na+/K+-ATPase inhibitor. Causes net influx of Ca2+; also initiates the rapidprotein kinase C–dependent inductions of early-response genes.Soluble in: WaterStock concentration: 100 mM (store at −20°C protected from light)Working concentration: 1 to 100 µMDuration of incubation: 30 min to 24 hr

PDMPUseful tool for studying the effects of cellular glycosphingolipid depletion. Blocksceramide glucosylation by inhibiting UDP-glucose:ceramide glucosyltransferase(glucosylceramide synthetase). Has antitumor activity; arrests 3T3 cells at both G1/Sand G2/M. Prevents sensitization of A431 cells to Shiga toxin. Slows the rate of bothanterograde vesicular traffic and endocytosis in CHO and BHK-21 cells. Redistrib-utes cis-Golgi proteins to the ER.Soluble in: EthanolStock concentration: 10 to 100 mM (store at 4°C)Working concentration: 20 to 100 µMDuration of incubation: 1 to 18 hr, depending on process studied

PD 98059 (MEK Inhibitor)Potent and selective inhibitor of MAP kinase kinase (MEK or MAPK/ERK kinase).Blocks the activity of MEK, thereby inhibiting the phosphorylation and activationof MAP kinase. Inhibits cell growth and reverses the phenotype of ras-transformed3T3 mouse fibroblasts and rat kidney cells. Inhibits Golgi reassembly in vitro.Cell-permeant.Soluble in: DMSOStock concentration: 50 mM (store at −20°C protected from light)Working concentration: 10 to 50 µMDuration of incubation: 30 min to 2 hr

PMSF (phenylmethanesulfonyl fluoride)Inhibits serine proteases like chymotrypsin, trypsin, and thrombin, as well asacetylcholinesterase and the cysteine protease papain (reversible by DTT treatment).PMSF inhibits serine proteases by sulfonating serine residues at the active site. Doesnot inhibit metalloproteases, most cysteine proteases, or aspartic proteases.Soluble in: Anhydrous isopropanol at 35 mg/ml with heating, resulting in a clear

to very slightly hazy, colorless to faint yellow solution, or in anhydrous(100%, not 95%) ethanol

Stock concentration: 17 mg/ml (store at room temperature)Working concentration: 17 to 170 µg/mlDuration of incubation: 15 min to 1 hr

PMSF is very unstable in the presence of water. The half-life of aqueous PMSF at 25°C atpH 7.0, 7.5, and 8.0 is 110, 55, and 35 min, respectively.

Pepstatin AInhibitor of aspartyl proteases, including pepsin, renin, cathepsin D, and HIV-1protease. Inhibits degradation of ApoB in rat hepatocytes; inhibits cytokine-inducedprogrammed cell death. Accelerates amyloid fibril formation in mice.Soluble in: DMSOStock concentration: 10 to 35 mM (store at −20°C)


A.1K.21


Working concentration: 50 to 100 µM in cellsDuration of incubation: 30 min to 2 hr

Pertussis toxinProtein endotoxin that catalyzes ADP-ribosylation of GDP-bound α subunits of theG proteins Gi, Go, and Gt. Uncouples G proteins from receptors, thereby keepingthe G protein in the inactive state. Used in the study of adenylate cyclase regulationand the role of Gi proteins. Consists of an enzymatically active A protomer subunit(S-1) which posesses both NAD+ glycohydrolase and ADP-ribosylation activitiesand a B oligomer subunit (S-2, S-3, S-4, and S-5) that is responsible for cell surfaceattachment.Stock solution: Reconstitute commercial preparation in water. Generally, commer-

cially available pertussis toxin (Alexis Biochemicals, Biomol, Calbiochem)contains 50 µg of protein in 10 mM sodium phosphate buffer, pH 7.0/50 mMsodium chloride after being resuspended in 0.5 ml water. It is an insoluble pro-tein that should be shaken gently before use. Store stock solutions at 4°C. Donot freeze.

Working concentration: 50 to 100 ng/mlDuration of incubation: 2 to 24 hr

Phenylarsine oxide (PAO)A cell-permeant phosphotyrosine phosphatase inhibitor (Ki = 18 µM). Induces adose-dependent increase in the free Ca2+ intracellular concentration in rat peritonealmacrophages, human foreskin fibroblasts, and cultured human endothelial cells,without affecting intracellular stores. Inhibits insulin activation of phosphatidyli-nositol 3′-kinase. Dithiol cross-linking agent.Soluble in: DMSO and chloroformStock concentration: 50 mM (store at room temperature)Working concentration: 10 to 50 µMDuration of incubation: Anywhere from 15 sec to 2 hr

Phorbol estersAn example is phorbol myristate acetate (PMA). Extremely potent tumor promoters.Activate protein kinase C by mimicking diacylglycerols (DAGs), causing a widerange of effects in cells and tissues.Soluble in: DMSOStock concentration: 1 mM (store at −20°C)Working concentration: 50 nM to 3 µMDuration of incubation: Cells can be incubated anywhere from 5 min to 48 hr;

stable in cells; results in long-term activation of PKC. However, long-termtreatment may cause down-regulation of certain PKC subtypes.

See Technical Notes #13 and #14 from Alexis Biochemicals for additional information.

PiceatannolAt low concentrations, inhibits the receptor-mediated activation of the proteintyrosine kinase Syk as compared to the Src family in mast cells and B cells. InhibitsFcεR1-mediated signaling in RBL-2H3 cells.Soluble in: DMSO, ethanolStock concentration: 10 to 50 mg/ml (store at 4°C protected from light)Working concentration: 10 to 30 µg/mlDuration of incubation: 1 hr


A.1K.22


Used inMolecular

Biology Research

PuromycinProtein synthesis inhibitor. Causes premature release of nascent polypeptide chainsby its addition to the growing chain end; structural analog of aminoacyl-tRNA.Soluble in: WaterStock concentration: 100 mM (store at −20°C)Working concentration: 10 to 100 µMDuration of incubation: 5 min to 1 hr

RapamycinMember of a family of macrolide immunosuppressants that binds to and inhibits thepeptidylproline cis-trans isomerase (PPIase) activity of the immunophilin FKBP12;effectors include a large protein termed FRAP (FKBP12 rapamycin-associatedprotein). FKBP12-rapamycin binds to but does not inhibit the activity of theCa2+/calmodulin–dependent serine/threonine phosphatase calcineurin. Blocks sig-naling, leading to the activation of p70 S6 kinase.Soluble in: DMSO, methanol, ethanolStock concentration: 2 mM (store at −20°C)Working concentration: 1 to 20 nMDuration of incubation: 30 min to 1 hr

Sodium azide (NaN3)Inhibits mitochondrial ATPases; generally used to deplete ATP levels within cells(often in combination with 50 mM 2-deoxyglucose).Soluble in: WaterStock concentration: 1 M (store at room temperature)Working concentration: 10 to 20 mMDuration of incubation: 15 to 90 min

Sodium butyrateA physiologically produced short-chain fatty acid that is generally used to increaseexpression of transfected genes with viral promoters (inhibits histone deacetyla-tion). Blocks serum-stimulated DNA synthesis via a G1 block. Induces apoptosis incolon carcinoma cell lines by a p53-independent process. Interferes with signal-transduction processes, including the release of Ca2+ from intracellular stores.Soluble in: WaterStock concentration: 5 M (store at –20o C)Working concentration: 2 to 5 mMDuration of incubation: Usually >12 hr

Sodium orthovanadateBroad-spectrum inhibitor of protein tyrosine phosphatases. Also inhibits otherATPases, by mimicking the γ phosphate of ATP, including Na+/K+ ATPase, acid andalkaline phosphatases, and adenylate cyclase. Vanadate is also a strong inhibitor oflysosomal proteolysis in hepatocytes, the effect being ascribed to a direct inhibitionof lysosomal enzymes. Stimulates pp60 (v-src) kinase activity in intact cells. It alsostimulates amino acid transport activity in skeletal muscle, in a rapid and concen-tration-dependent manner.Simple aqueous solutions of (VO4)

3− ion involve a dozen or more ionic species, bothmonomeric and oligomeric, whose abundances depend upon pH and [VO4]

3− con-centration. See Fohr et al. (1989) for directions on preparing monomeric or-thovanadate. It is unclear how readily vanadate ions enter cells (likely through aniontransporters). At the concentration required for maximum inhibition, vanadate mayhave side effects that limit its application in cell culture. Can be combined with

continued


A.1K.23


hydrogen peroxide (forming peroxyvanadate) to facilitate cell entry (combine 100µl 0.1 M orthovanadate, 900 µl water, and 3.3 µl 30% H2O2; use 1:100 dilution ofthis on cells). However, the effect of hydrogen peroxide itself should be tested.Soluble in: Water Stock concentration: 100 mM (store at room temperature). To ensure the presenceof monomers, the solution is heated to boiling until translucent and the pH isreadjusted to 10. Solutions can be divided into aliquots, stored in plastic, and frozen.The orange color observed before boiling is due to decavanadate. At pH 10 this willslowly depolymerize over several hours to the colorless monovanadate. Vanadyl,metavanadate, orthovanadate, and decavanadate will interconvert in aqueous solu-tion without suitable precautions (i.e., control of pH, oxidation state, complexingcompounds, and concentration).Working concentration: 200 µM to 2 mMDuration of incubation: 15 min to 2 hr

StaurosporineA potent cell-permeant inhibitor of protein kinases, most potently protein kinase C(Ki = 0.7 nM), protein kinase A (Ki = 7 nM), and myosin light chain kinase (Ki = 1.3nM). Interaction is with the ATP binding site. Induces apoptosis, but not DNAfragmentation in MCF-7 cells. Arrests normal cells at the G1 checkpoint.Soluble in: DMSO, methanolStock concentration: 1 mM (store at −20°C protected from light)Working concentration: 10 to 200 nMDuration of incubation: 30 min to 24 hr, depending on the assay used

See Table I (Selectivity of Selected Kinase Inhibitors) in the Biomol Catalog and Handbook,5th ed.

Taxol (Paclitaxel)Antitumor and antileukemic agent. Promotes assembly of microtubules and inhibitsmicrotubule disassembly. Bundles microtubules after several hours. Similar tonocodazole, taxol can inhibit microtubule dynamics without affecting overall poly-mer levels at nanomolar concentrations. Blocks cells at the G2/M stage. Inducesapoptosis in several cell types.Soluble in: DMSO, methanolStock concentration: 20 mM (store at −20°C protected from light)Working concentration: 10 nM to 20 µMDuration of incubation: Taxol works rapidly to stabilize microtubules (within sev-

eral minutes), although bundling takes several hours (this may be facilitated byfirst depolymerizing the polymer pool with ice treatment and/or washout oflow levels of nocodazole)

ThapsigarginPotent inhibitor of sarcoplasmic reticulum (SR)/endoplasmic reticulum (ER) Ca2+-ATPases. Induces IP3-independent release of Ca2+ from the endoplasmic reticulum,causing an increase in intracellular Ca2+. Depletion of Ca2+ from intracellular storesinduces stress response and defects in protein folding and processing. Inducesapoptosis in rat thymocytes and in human hepatoma cells. Irreversible.Soluble in: DMSO, ethanolStock concentration: 1 mM (store at −20°C in aliquots, protect from light)Working concentration: 20 nM to 1 µMDuration of incubation: 15 sec to 2 min produces rise in intracellular Ca2+;

longer incubations may be used, depending on effect to be analyzed



A.1K.24


Used inMolecular

Biology Research

TrifluoperazineCalmodulin antagonist. At 10 µM, potentiates rise in cytosolic calcium induced byagonists. Antagonizes calmodulin at higher concentrations. Inhibits IL-2 productionin activated Jurkat T cells. Structurally distinct from W-7.Soluble in: Water (dihydrochloride salt)Stock concentration: 10 mM (store at 4°C)Working concentration: 10 to 50 µMDuration of incubation: 10 min to 3 hr

TunicamycinNucleoside antibiotic that inhibits N-linked glycosylation, specifically by blockingthe transfer of N-acetylglucosamine-1-phosphate from UDP-N-acetylglucosamineto dolichol monophosphate; has no effect on other glycosylation forms, such asSer/Thr-linked oligosaccharides. Causes misfolding and retention of numerousglycoproteins in the endoplasmic reticulum, which induces synthesis of ERchaperones.Soluble in: DMSO, ethanolStock concentration: 10 mg/ml (store at −20°C)Working concentration: 1 to 10 µg/mlDuration of incubation: Cells can be treated from 1 to 24 hr

TyrphostinsLarge family of protein tyrosine kinase inhibitors. Inhibits receptors such as EGFreceptor and PDGF receptor.Soluble in: DMSO, ethanolStock concentration: 20 to 100 mM (store at −20°C protected from light)Working concentration: 10 to 150 µMDuration of incubation: Anywhere from 1 to 48 hr

See Technical Note #22 from Alexis Biochemicals for additional information. Also see TableII (Selectivity of Tyrosine Protein Kinase Inhibitors) in the Biomol Catalog and Handbook,5th ed.

ValinomycinPotassium ionophore. Decreases ATP synthesis by decreasing membrane potentialat mitochondrial membranes. Reported to inhibit NGF-induced neuronal differen-tiation. Used with nigericin to adjust cytoplasmic pH.Soluble in: DMSOStock concentration: 1 mM (store at room temperature)Working concentration: 1 to 20 µMDuration of incubation: Normally 15 min to 2 hr

VinblastineVinca alkaloid; antitumor drug. Inhibitor of cell proliferation that acts by disruptingspindle microtubule function. Binds tubulin and suppresses microtubule dynamics.Depolymerizes microtubules at higher concentrations. Induces apoptosis in culturedhepatocytes and human lymphoma cells. Similar, but not identical effects observedwith another vinca alkaloid, vincristine.Soluble in: MethanolStock concentration: 20 mM (store at 4°C protected from light)Working concentration: <10 nM (suppresses microtubule dynamics); 100 nM to

1 µM (depolymerizes microtubules); >10 µM (forms non-microtubule poly-mers)

Duration of incubation: 30 min to 24 hr


A.1K.25


W-7Member of a family of calmodulin antagonists, inhibiting calcium/calmodulinregulated enzyme activity. W-7 inhibits the Ca2+/calmodulin–induced activation ofmyosin light chain kinase (Ki = 51 µM) and phosphodiesterase (Ki = 28 µM). Inhibitsmembrane tubulation in cells treated with brefeldin A.Soluble in: WaterStock concentration: 10 to 100 mM (store at 4°C protected from light)Working concentration: 10 to 100 µMDuration of incubation: 30 min to 2 hr

WortmanninSelective and potent phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor; formscovalent associations with the kinases and is, therefore, irreversible. AbolishesPDGF-mediated Ins(3,4,5)P3 formation in fibroblasts. Blocks the metabolic effectsof insulin in isolated rat adipocytes without affecting the insulin receptor tyrosinekinase activity. Inhibits the formation of constitutive transport vesicles from theTGN. In human fetal undifferentiated cells, wortmannin induces morphological andfunctional endocrine differentiation.Soluble in: DMSOStock concentration: 1 to 20 mM (store at −20°C in aliquots, protected from light)Working concentration: 10 to 100 nMDuration of incubation: 30 min to 4 hr

At nanomolar concentrations, wortmannin is specific to PI 3-kinases, while at higherconcentrations other kinases are affected. Once diluted into aqueous solutions, wortmanninis less stable and should be made fresh daily.

LITERATURE CITED

Fohr, K.J., Scott, J., Ahnert-Hilger, G., and Gratzl, M. 1989. Characterization of the inositol 1,4,5-trisphos-phate-induced calcium release from permeabilized endocrine cells and its inhibition by decavanadate andp-hydroxymercuribenzoate. Biochem. J. 262:83-89.

Jalink, K., van Corven, E.J., and Moolenaar, W.H. 1990. Lysophosphatidic acid, but not phosphatidic acid,is a potent Ca2+-mobilizing stimulus for fibroblasts. Evidence for an extracellular site of action. J. Biol.Chem. 265:12232-12239.

Seufferlein, T. and Rozengurt, E. 1994. Lysophosphatidic acid stimulates tyrosine phosphorylation of focaladhesion kinase, paxillin, and p130: Signaling pathways and cross-talk with platelet-derived growth factor.J. Biol. Chem. 269: 9345-9351.

Contributed by Nelson B. ColeUniversity of PennsylvaniaPhiladelphia, Pennsylvania


A.1K.26


Used inMolecular

Biology Research

Documents

STANDARD MEASUREMENTS, DATA, Common Abbreviations