Living Colors User Manual - Home | Perelman School of ......Clontech Laboratories, Inc. Protocol No. PT2040-1 4 Version No. PR1Y691 Living Colors® User Manual • Red Fluorescent

Living Colors®

User Manual

PT2040-1 (PR1Y691)Published 26 November 2001

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT2040-1 2 Version No. PR1Y691

Living Colors® User Manual

I. Introduction 3

II. Properties of GFP and GFP Variants 5

III. Expression of GFP and GFP Variants 14

IV. Detection of GFP, GFP Variants, and DsRed 19

V.Purified Recombinant GFP and GFP Variants 28

VI. GFP Antibodies 30

VII. Troubleshooting Guide 32

VIII. References 36

IX. Related Products 42

Appendix: Recommended Resources & Reading 46

List of Figures

Figure1. GenealogyofGFPproteins. 4

Figure2. AminoacidsequencedifferencesbetweenwtGFP anditsvariants. 5

Figure3. ExcitationandemissionspectraofwtGFPanda red-shiftedGFPexcitationvariant(GFP-S65T). 6

Figure4. DsRed,EGFP,ECFP,andEYFPhavedistinctabsorptionandemissionspectra. 7

Figure5. Flowcytometryanalysisofd2EGFPstability. 9

Figure6. ComparisonoffluorescenceintensityofGFPinsonicationbufferversusGFPincelllysates. 25

Figure7. Relativefluorescenceintensityoftwo-foldserialdilutionsofasuspensionofGFP-transformedcellsinsonicationbuffer. 27

List of Tables

TableI. LivingColors®FluorescentProteins:SpectralProperties 13

TableII. PartialListofProteinsExpressedasFusionstoGFP 15–16

TableIII. MulticoloranalysiswithLivingColors®Proteins 21

Table of Contents

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Thebioluminescent jellyfishAequoreavictoriaproduces lightwhenenergy istransferredfromCa2+-activatedphotoproteinaequorintogreenfluorescentpro-tein(GFP;Shimomuraetal.,1962;Morin&Hastings,1971;Wardetal.,1980).Thecloningofthewild-typeGFPgene(wtGFP;Prasheretal.,1992;Inouye&Tsuji,1994a)anditssubsequentexpressioninheterologoussystems(Chalfieetal.,1994;Inouye&Tsuji,1994a;Wang&Hazelrigg,1994)establishedGFPas a novel genetic reporter system. When expressed in either eukaryotic orprokaryoticcellsandilluminatedbyblueorUVlight,GFPyieldsabrightgreenfluorescence.Light-stimulatedGFPfluorescence is species-independentanddoesnotrequireanycofactors,substrates,oradditionalgeneproductsfromA.victoria.Additionally,detectionofGFPanditsvariantscanbeperformedinlivingcellsandtissuesaswellasfixedsamples.

ClontechmakesGFPandGFPvariantsavailableforresearchpurposesthroughitslineofLiving Colors®Fluorescent Proteins (Figure1).LivingColorsproteinsareexcellentreportersforgeneexpressionandproteinlocalizationstudies.Fol-lowingisalistoftheLivingColorsGFPvariantscurrentlyoffered.Youwillfindthatthesevariantsarewellsuitedforfluorescencemicroscopyandflowcytometry.

•GFPexcitationvariants: – EGFP, enhanced green fluorescent protein (GFPmut1; Cormack et

al.,1996),anddestabilized EGFP (dEGFP;Lietal.,1998)havered-shiftedexcitationspectra.Whenexcitedwithblue light, theyfluoresce4–35-foldmorebrightlythanwtGFP.

•GFPemissionvariants: – EYFP (enhanced yellow fluorescent protein) contains four amino acid

substitutionsthatshifttheemissionfromgreentoyellowish-green.ThefluorescencelevelofEYFPisroughlyequivalenttothatofEGFP.

– ECFP(enhancedcyanfluorescentprotein)containssixaminoacidsub-stitutions,oneofwhichshiftstheemissionspectrumfromgreentocyan(Heim&Tsien,1996;Miyawakietal.,1997).Itisasuitabledonormoleculeinfluorescenceresonanceenergy transfer(FRET)studiesandcanbeusedfordual-labelingfluorescencemicroscopyinconjunctionwithEYFP(Greenetal.,2000).

•UV-OptimizedGFPvariant: – GFPuvisreportedtobe18timesbrighterthanwtGFPwhenexpressed

inE.coliandexcitedbystandardUVlight(Cramerietal.,1996).ThisvariantcontainsadditionalaminoacidmutationswhichalsoincreasethetranslationalefficiencyoftheproteininE.coli.

I. Introduction



•RedFluorescentProtein: – WealsoofferDsRed2,aredfluorescentproteinthatderivesfromacoral

oftheDiscosomagenus,referredtoasDiscosomasp.(Ds).Becauseofitsdistinctredemission,DsRed2isaperfectcomplementtothegreen,yellow,andcyanfluorophores.WiththedevelopmentoffiltersdesignedspecificallyfordetectingEGFP,EYFP,ECFP,andDsRed,it’snowpos-sibletousethesereportersincombinationtocarryouttwo-,three-,or,even,four-coloranalyses.TheRedFluorescentProtein,DsRed,ismorecarefullydescribedinaseparateUserManual,PT3404-1,whichcanbedownloadedfromourwebsiteatwww.clontech.com.

Inadditiontotheirchromophoremutations,theenhancedGFPvariantgenesEGFP,EYFP,andECFPcontainmorethan190silentmutationsthatcreateanopenreadingframecomprisedalmostentirelyofpreferredhumancodons(Haas,etal.,1996).Furthermore,upstreamsequencesflankingthecodingregionshavebeenconvertedtoaKozakconsensustranslationinitiationsite(Kozak,1987),andpotentiallyinhibitoryflankingsequencesintheoriginalcDNAclones(lgfp10&pGFP10.1;Chalfieetal.,1994)wereremoved.AllofthesechangesenhancethetranslationalefficiencyofthemRNA—and,consequently,theexpressionoftheGFPvariant—inmammalianandplantsystems.

I. Introduction continued

Figure 1. Genealogy of GFP proteins. GFPmut1(Cormacketal.,1996),thered-shiftedvariantusedtocreatethevariantsshown,isnotsoldbyClontech.

GFPuvGFPmut1

GFP (wt)

EGFP

d2EGFPd1EGFP d4EGFPEYFP ECFP

d2EYFP d2ECFP



II. Properties of GFP and GFP Variants

A. The GFP Chromophore: Structure and Biosynthesis TheGFPchromophoreconsistsofacyclic tripeptidederived fromSer-

Tyr-Gly(aminoacids65–67)intheprimaryproteinsequence(Codyetal.,1993)andisonlyfluorescentwhenembeddedwithinthecompleteGFPprotein.Ofthecomplete238-amino-acidpolypeptide,aminoacids7–229arerequiredforfluorescence(Lietal.,1997).ThecrystalstructureofGFPhasrevealedatightlypackedβ-canenclosinganα-helixcontainingthechro-mophore(Ormöetal.,1996;Yang,F.etal.,1996).Thisstructureprovidestheproperenvironmentforthechromophoretofluoresce.NascentGFPisnotfluorescent,sincechromophoreformationoccurspost-translationally.Thechromophoreisformedbyacyclizationreactionandanoxidationstepthatrequiresmolecularoxygen(Heimetal.,1994;Davis,D.F.etal.,1995).Thesestepsareeitherautocatalyticorusefactorsthatareubiquitous,sincefluorescentGFPformsinabroadrangeoforganisms.Chromophoreforma-tionmaybetherate-limitingstepformaturationofthefluorescentprotein(Heimetal.,1994;Davis,D.F.etal.,1995).Wild-typeGFPabsorbsUVandbluelightandemitsgreenlight.

B. GFP Excitation Variants Thered-shiftedvariantsEGFPanddEGFP(destabilizedforrapidturnover)

containmutationsinthechromophore(Figure2)whichshiftthemaximalexcitationpeakto~490nm(Figure3).

wt GFP:

EGFP:

EYFP:

ECFP:

Phe 64 Ser Tyr Gly Val Gln 69 . . . Ser72 . . .Tyr 145 Asn146. . . Met153. . . Val163. . . Thr203

Leu 64 Thr Tyr Gly Val Gln 69

Phe64 Gly Tyr Gly Leu Gln69 . . . Ala72. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .Tyr203

Leu 64 Thr Trp Gly Val Gln 69. . . . . . . . . . . . . . . . . Ile146. . . . Thr153. . . . Ala163

Figure 2. Amino acid sequence differences between wt GFP and its variants. Aminoacidsub-stitutionsareunderlined.

The major excitation peak of the red-shifted variants encompasses theexcitationwavelengthofcommonlyusedfiltersets.Similarly,theargon-ionlaserused inmostFACSmachinesandconfocalscanning lasermicro-scopesemitsat488nm,soexcitationofthered-shiftedGFPvariantsismuchmoreefficientthanexcitationofwtGFP.Inpracticalterms,thismeansthedetectionlimitsinbothmicroscopyandFACSareconsiderablylowerwiththered-shiftedvariants.Dual-labelingexperimentscanbeperformedbyselectiveexcitationofwtGFPandred-shiftedGFP(Kainetal.,1995;Yang,T.T.etal.,1996b).ItisalsopossibletouseEGFPandGFPuvindouble-labelingexperiments.



II. Properties of GFP and GFP Variants continued

• EGFP contains two amino acid substitutions: F64L and S65T(Figure2).Basedonspectralanalysis,EGFPfluoresces35-foldmoreintenselythanwtGFPwhenexcitedat488nm(Cormacketal.,1996;Yang,T.T.etal.,1996a)duetoanincreaseinitsextinctioncoefficient(Em;TableI)andahigherefficiencyofchromophoreformation.EGFPchromophoreformationismuchmoreefficientat37°CthanwtGFP;95% of the soluble EGFP protein contains the chromophore whenexpressedatthistemperature(Pattersonetal.,1997).

C. GFP Emission Variants • Cyan Emission Variant (ECFP) TheECFPvariantcontainssixaminoacidsubstitutions(Figure2).One

of these,Tyr-66 toTrp, shifts thechromophore’sexcitationmaximato433nm(majorpeak)and453nm(minorpeak),andtheemissionmaximato475nmwithasmallshoulderat501nm(TableI;Figure4).TheotherfivesubstitutionsenhancethebrightnessandsolubilityoftheproteininamannersimilartotheotherEGFPvariants.

• Yellow Emission Variant (EYFP) The EYFP variant contains four different amino acid substitutions

(Figure2)thatshiftthefluorescencefromgreen(509nm)toyellow-green(527nm;TableI;Figure4).AlthoughEYFP’sfluorescenceexcitationmaximumis513nm,itcanbeefficientlyexcitedat488nm,thestandardlaserlineforanargon-ionlaser.

Figure 3. Excitation and emission spectra of wt GFP and a red-shifted GFP excitation variant (GFP-S65T).Excitation:dashedlines.Emission:solidlines.TheemissiondataforwtGFPwereob-tainedbyexcitingtheproteinat475nm.(TheemissionpeakforwtGFPisseveral-foldhigherwhenilluminatedat395nm).TheemissiondataforGFP-S65Twereobtainedbyexcitingtheproteinat489nm(Heimetal.,1995).EGFPandGFP-S65Thavesimilarspectra.

400 500Wavelength (nm)

Abs

orba

nce

or R

elat

ive

Fluo

resc

ence

GFP-S6�T

wt GFP

wt GFP

GFP-S6�T




Figure 4. DsRed, EGFP, ECFP, and EYFP have distinct absorption and emission spectra. Panel A.Absorbance.Panel B.Emission.Curvesarenormalized;theheightofeachcurveisnotanindicationofrelativesignalstrengthperfluorophore.EGFP,ECFP,andEYFPareallvariantsofthegreenfluorescentproteinfromAequoreavictoria,denotedasAvinthefigure.DsRedisderivedfromacoraloftheDiscosomagenusreferredtoasDiscosomasp.(Ds).(SpectraforEGFP,ECFP,andEYFPwereprovidedbyD.W.Piston,VanderbiltUniversity.)

A

B

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375325 425 475 525 550 575 600

Wavelength (nm)

Nor

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abs

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AvEGFP

AvECFP

AvEYFP DsRed

350 400 450 500

0

20

40

60

80

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375325 425 475 525 550 575 600

Wavelength (nm)

Nor

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abs

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AvEGFP

AvECFP

AvEYFP DsRed

350 400 450 500

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D. UV-Optimized GFP Variant GFPuv isoptimized formaximalfluorescencewhenexcitedbyUV light

(360–400 nm) and for higher bacterial expression. The GFPuv variantis idealforexperimentsinwhichGFPexpressionwillbedetectedusingUVlightforchromophoreexcitation(e.g.,forvisualizingbacteriaoryeastcolonies).GFPuvwasdevelopedbyCramerietal.(1996).

GFPuvcontainsthreeaminoacidsubstitutions(Phe-99toSer,Met-153toThr,andVal-163toAla[basedontheaminoacidnumberingofwtGFP]),noneofwhichalter thechromophoresequence.ThesemutationsmakeE.coliexpressingGFPuvfluoresce18timesbrighterthanwtGFP(Cramerietal.,1996).WhilethesemutationsdramaticallyincreasethefluorescenceofGFPuvthroughtheireffectsonproteinfoldingandchromophoreformation,theemissionandexcitationmaximaremainatthesamewavelengthsasthoseofwtGFP(Cramerietal.,1996;Figure3;TableI).However,GFPuvhasagreaterpropensitytodimerizethanwtGFP.

GFPuvexpressedinE.coli isasoluble,fluorescentproteinevenunderconditionsinwhichthemajorityofwtGFPisexpressedinanonfluorescentformininclusionbodies.ThisGFPvariantalsoappearstohavelowertoxicitythanwtGFP;hence,theE.colicontainingGFPuvgrowtwotothreetimesfasterthanthoseexpressingwtGFP(Cramerietal.,1996).Furthermore,theGFPuvgene isasyntheticGFPgene inwhichfive rarelyusedArgcodons from thewtgenewere replacedbycodonspreferred inE.coli.Consequently,theGFPuvgeneisexpressedveryefficientlyinE.coli.

E. Destabilized GFP Variants Inmammaliancells,destabilizedvariants(dEGFP,dECFP,anddEYFP)aredegradedmorerapidlythanwtGFP.Inordertocreatethedestabilizedvari-ants,EGFP,ECFP,andEYFPwerefusedtoaminoacidresidues422–461ofmouseornithinedecarboxylase(MODC).ThisC-terminalregionofMODCcontainsaPEST(Pro-Glu-Ser-Thr)aminoacidsequencethattargetstheproteinforrapidintracellulardegradation.Thed1,d2,andd4variantsex-hibithalf-livesof1,2,or4hours,respectively,comparedtoEGFP,ECFP,andEYFP,whichhavehalf-livesof>24hours.Half-livesweredeterminedbyflowcytometry(Figure5),fluorescencemicroscopy,andWesternblotanalysis(Lietal.,1998;July1999&April1998Clontechniques).

Thefluorescenceintensityandspectralpropertiesofdestabilizedvariantsare identical to thoseof theoriginal chromophore,butbecauseof theirshortenedhalf-lives,destabilizedvariantscanbeusedtomoreaccuratelymeasurethekineticsofpromoteractivityorthetransientexpressionofaproteintowhichtheyarefused.

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Time after CHX addition (hr)

EGFP

d2EGFP

% F

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nt c

ells

0.00

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1.0 2.0 3.0

Figure 5. Flow cytometry analysis of d2EGFP stability. CHO-K1Tet-Off™cellsweretransfectedwithpTRE-EGFPorpTRE-d2EGFP.After24hr,thetransfectedcellsweretreatedwith100µg/mlcycloheximide(CHX)for0,1,2,or3hrtoinhibitproteinsynthesis.TheCHX-treatedcellswerehar-vestedwithEDTA,and1x105cellswereusedforflowcytometryanalysis.Thepercentagesofcellswithfluorescenceabovebackgroundareplottedonthey-axis.Valuesarenormalizedtofluorescencelevelsatthezerotimepoint,definedas100%.After3hr,fluorescenceintensityofthecellpopulationexpressingd2EGFPwasreducedto30%ofinitialintensity.

F. Acquisition and Stability of GFP Fluorescence 1. Protein solubility Inbacterialexpressionsystems,muchoftheexpressedwtGFPisfound

inan insoluble,nonfluorescent formin inclusionbodies. Incontrast,almostallEGFPandGFPuv isexpressedasasoluble,fluorescentprotein.

2. Photobleaching GFPfluorescenceisverystableinafluorometer(Ward,pers.comm.).

Even under the high-intensity illumination of a fluorescence micro-scope,GFP ismore resistant tophotobleaching than is fluorescein(Wang&Hazelrigg,1994;Niswenderetal.,1995).ThefluorescenceofwtGFPandEGFPisquitestablewhenilluminatedwith450–490nmlight(themajorexcitationpeakforthered-shiftedexcitationvariants,buttheminorpeakforwtGFP).SomephotobleachingoccurswhenwtGFPisilluminatednearitsmajorexcitationpeakwith340–390nmor395–440nmlight(Chalfieetal.,1994;Niswenderetal.,1995).TherateofphotobleachingofwtGFPand itsgreenvariantsalsovarieswith theorganismbeing studied; for example,GFPfluorescence isquitestableinDrosophila(Wang&Hazelrigg,1994)andzebrafish.InC.elegans,10mMNaN3acceleratesphotobleaching(Chalfieetal.,1994).StudiesofthephotobleachingcharacteristicsofGFPuvhavenotbeenperformedtodate.




WhenusingECFP,photobleachingdoesnotposeamajorproblem,andwerecommendusingthisvariantifphotobleachinginterfereswith your studies. (Rapid photobleaching has been a problemwithpreviouslydescribedbluevariantsofGFP.)However,ifyoudecidetofixandmountyourspecimens,commerciallyavailablemountingsolutionsthatdonotcontainantibleachingreagentscancauseseverephotobleachingwithinsecondswhentheslidesareexposedtolightduringfluorescencemicroscopy.Besuretokeepmountedslidesinthedarkat4°C.

3. Stability to oxidation/reduction GFPneedstobeinanoxidizedstatetofluorescebecausechromo-

phoreformationisdependentuponanoxidationofTyr-66(Heimetal.,1994).Strongreducingagents,suchas5mMNa2S2O4or2mMFeSO4,convertGFPintoanonfluorescentform,butfluorescenceisfullyrecoveredafterexposuretoatmosphericoxygen(Inouye&Tsuji,1994b).Weakerreducingagents,suchas2%β-mercaptoethanol,10mMdithiothreitol(DTT),10mMreducedglutathione,or10mML-cysteine(Inouye&Tsuji,1994b),ormoderateoxidizingagents(Ward,pers.comm.),donotaffectthefluorescenceofGFP.

4. Stability to pH wtGFP retains fluorescence in the rangepH5.5–12; however,

fluorescenceintensitydecreasesbetweenpH5.5andpH4,anddropssharplyabovepH12(Bokman&Ward,1981).EGFPexhib-itsareducedrangeofpHstability.Forthisvariant,fluorescenceisstablebetweenpH7.0andpH11.5,dropssharplyabovepH11.5,anddecreasesbetweenpH7.0andpH4.5,retainingabout50%offluorescenceatpH6.0(Pattersonetal.,1997;Ward,pers.comm.).BecauseGFPissosensitivetopH,itcanbeusedasanintracellularpHindicatorinlivingcells(Kneenetal.,1998).

5.Stability to chemical reagents GFPretainsitsfluorescenceinmilddenaturants,suchas1%SDS

or8Murea,andafterfixationwithglutaraldehydeorformaldehyde.FullydenaturedGFPisnotfluorescent.GFPissensitivetosomenailpolishesusedtosealcoverslips(Chalfieetal.,1994;Wang&Hazelrigg,1994);therefore,usemoltenagaroseorrubberce-menttosealcoverslipsonmicroscopeslides.Fluorescenceisalsoquenchedbythenematodeanestheticphenoxypropanol(Chalfieetal., 1994).GFPfluorescence is irreversiblydestroyedby1%H2O2andsulfhydryl reagentssuchas1mMDTNB (5,5'-dithio-bis-[2-nitrobenzoicacid]) (Inouye&Tsuji,1994b).Manyorganicsolventscanbeusedatmoderateconcentrationswithoutabolishingfluorescence;however,theabsorptionmaximummayshift.




GFPisnonfluorescentinabsoluteethanolandisunlikelytowithstandthecompletedehydrationrequiredforparaffinorplasticembedding.

6. Protein stability in vitro: GFP is exceptionally resistant to heat (Tm=70°C), alkaline pH,

detergents,chaotropicsalts,organicsolvents,andmostcommonpro-teases,exceptpronase(Bokman&Ward,1981;Ward,1981).SomeGFPfluorescencecanbeobservedwhennanogramamountsofproteinareresolvedonnativeor1%SDSpolyacrylamidegels(Inouye&Tsuji,1994a).

FluorescenceislostifGFPisdenaturedbyhightemperature,pHex-tremes,orguanidiniumchloride,butcanbepartiallyrecoverediftheproteinisallowedtorenature(Bokman&Ward,1981;Ward&Bokman,1982).Athiolcompoundmaybenecessarytorenaturetheproteinintothefluorescentform(Surpin&Ward,1989).

in vivo: GFPappearstobestablewhenexpressedinvariousorganisms;EGFP

hasanestimatedhalf-lifeof>24hours(Lietal.,1998). 7. Temperature sensitivity of GFP chromophore formation AlthoughfluorescentGFPishighly thermostable(seeabove), itap-

pears that the formation of the GFP chromophore is temperaturesensitive. Inyeast,GFPfluorescencewasstrongestwhen thecellsweregrownat15°C,decreasingtoabout25%ofthisvalueasthein-cubationtemperaturewasraisedto37°C(Limetal.,1995).However,GFPandGFP-fusionproteins synthesized inS. cerevisiaeat 23°Cretain fluorescence despite a later shift to 35°C (Lim et al., 1995).It has also been noted that E. coli expressing GFP show strongerfluorescencewhengrownat24°Cor30°Ccomparedto37°C(Heimetal.,1994;Ward,pers.comm.).MammaliancellsexpressingGFPhavealsobeenseentoexhibitstrongerfluorescencewhengrownat30–33°Ccomparedto37°C(Pines,1995;Ogawaetal.,1995).

AlltheenhancedGFPvariantsshowlittledifferenceinfluorescencewhenexpressedateither25°Cor37°C.Themutationsthatincreasetheefficiencyofproteinfoldingandchromophoreformation(Cormacketal.,1996,Cramerietal.,1996)alsosuppressthethermosensitivityofchromophoreformation.

8. Dimerization of GFP GFPisfluorescenteitherasamonomerorasadimer.Theratioof

monomericanddimericformsdependsontheproteinconcentrationandenvironment.wtGFPdimerizesviahydrophobic interactionsatproteinconcentrationsabove5–10mg/mlandinhigh-saltconditions.




Dimerizationresultsina4-foldreductionintheabsorbanceat470nmanda concomitant increase inabsorbanceat 395nm (Ward, pers.comm.).GFPuvhasagreaterpropensitytodimerizethanwtGFP;the470-nmexcitationpeakissuppressedatproteinconcentrationsabout5-foldlowerthanforwtGFP(Ward,pers.comm.).InmostexperimentalsystemsusingwtGFPasareporter,themonomericformwillpredomi-nate;however,whenGFPuvisusedasthereporter,dimerizationmaybeevidentevenatmoderateexpressionlevels.

G. Sensitivity and fluorescence intensity 1. General considerations GFPfusionproteinshavebeenfoundtogivegreatersensitivityand

resolutionthanstainingwithfluorescentlylabeledantibodies(Wang&Hazelrigg,1994).GFPfusionsaremoreresistanttophotobleachingandexhibitlowbackgroundfluorescence(Wang&Hazelrigg,1994).

Because GFPuv, EGFP, and dEGFP exhibit higher extinction coef-ficients thanwtGFP, theyprovide significantly increased sensitivityasreportermolecules.Furthermore,subcellularlocalizationofEGFP(e.g.,tothecytoskeleton)canproduceanevenmoreintensesignalbyconcentratingthesignaltoaspecificareawithinthecell.However,forsomeapplications,thesensitivityofGFPmaybelimitedbyautofluo-rescenceorlimitedpenetrationoflight.StudieswithwtGFPexpressedinHeLacells(Niswenderetal.,1995)haveshownthatthecytoplasmicconcentrationmustbegreaterthan~1.0µMtoobtainasignalthatistwicetheautofluorescence.Thisthresholdfordetectionislowerwiththe red-shifted excitation variant: ~100 nM for EGFP (Piston, pers.comm.).

2. As a quantitative reporter Thesignal fromGFPand itsvariantsdoesnothaveanyenzymatic

amplification;hence,thesensitivityofGFPwillprobablybelowerthanthatforenzymaticreporterssuchasβ-galactosidase,SEAP,andfireflyluciferase.However,GFPsignalscanbequantifiedbyflowcytometry,confocalscanninglasermicroscopy,andfluorometricassays.PurifiedGFPandGFPvariantscanbequantifiedinafluorometer-basedassayinthelow-nanogramrange.Furthermore,usingdestabilizedGFPsasreporterscanincreasesensitivitybypreventingaccumulationofGFPunderbasalornon-inducedexpression.Therefore,destabilizedGFPseffectivelyextendtheinductionwindowbyincreasingfold-induction.

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TABLE I. LIVInG COLORS® FLUORESCEnT PROTEInS: SPECTRAL PROPERTIES

Protein Excitation Emission Em (cm–1M–1) Quantum Yield (%) Maxima (nm) Maxima (nm)

wtGFP 395(470) 509(540) 9,500a ~80a

EGFPd 488 507 55,000b ~60b

ECFPd 433(453) 475(501) 26,000b ~40b

EYFPd,e 513 527 84,000b ~61b

GFPuv 395 509

DsRede 558 583 75,000c ~70c

aPattersonetal.,1997bDavePiston,pers.comm.c Bairdetal.,2000;SlightlylowervaluesarereportedbyPattersonetal.,2001.dThedestabilizedvariant(dEGFP,dECFP,ordEYFP)hasidenticalexcitationandemission

maxima,butashorterhalf-life.eEYFPandDsRedcanalsobeexcitedat488nm.SeeFigure4.



III. Expression of GFP and GFP Variants continued

A. Suitable Host Organisms and Cells GFP fluorescence is species-independent. Fluorescence has been

reportedfrommanydifferenttypesofGFP-expressinghosts.BelowisapartiallistingoforganismsthatcanexpressfluorescentGFP;amoreex-tensivelistingwithreferencescanbefoundatwww.clontech.com/gfp.

1. Microbes Agrobacteriumtumefaciens Myxococcusxanthus Anabaena Polysphondyliumpallidum Bacillussubtilis Pseudomonas Bartonellahenselae Rhizobiummeloliti Candidaalbicans Saccharomycescerevisiae Caulobactercrescentus Salmonella Dictyostelium Schizosaccharomycespombe Escherichiacoli Ustilagomaydis Helicobacterpylori Yersinia Mycobacterium

2. Invertebrates Aedesaegypti Caenorhabditiselegans Diamondbackmoth Drosophilamelanogaster Lytechinuspictus

3. Vertebrates Fish, avian,andmammaliancell lines (specific cell lines listedon

web) Mouse Xenopus Zebrafish

4. Plants Arabidopsisthaliana(thalecress) Citrussinensis(L.)Osbeckcv.Hamlin(anembryogenicsweet-orange

cellline,H89) Glycinemax(soybean) Hordeumvulgar(barley) Nicotianabenthamiana&Nicotianaclevelandii(tobacco) Onion Zeamays(maize)




B. Expression of GFP Fusion Proteins GFPhasbeenexpressedasafusiontomanydifferentproteins(TableII).

Inmanycases,chimericgenesencodingeitherN-orC-terminalfusionstoGFPretainthenormalbiologicalactivityoftheheterologouspartner,aswellasmaintainingfluorescentpropertiessimilartonativeGFP(Flachetal.,1994;Wang&Hazelrigg,1994;Marshalletal.,1995;Stearns,1995).TheuseofGFPanditsvariantsinthiscapacityprovidesa“fluorescenttag”ontheprotein,whichallowsforinvivolocalizationofthefusionprotein.GFPfusionscanprovideenhancedsensitivityandresolutionincomparisontostandardantibodystainingtechniques(Wang&Hazelrigg,1994),andtheGFPtageliminatestheneedforfixation,cellpermeabilization,andanti-bodyincubationstepsnormallyrequiredwhenusingantibodiestaggedwithchemicalfluorophores.Lastly,useoftheGFPtagpermitskineticstudiesofproteinlocalizationandtrafficking(Flachetal.,1994;Wang&Hazelrigg,1994).

TABLE II. PARTIAL LIST OF PROTEInS EXPRESSED AS FUSIOnS TO GFP

Host Cell orProtein Organism Localization References

CotE Bacillussubtilis Forespore Webbetal.,1995

DacF,SpoIVA Bacillussubtilis Prespore/mothercell Lewis&Errington,1996

Coronin D.discoideum Phagocyticcups Maniaketal.,1995

Myosin D.discoideum Mooresetal.,1996

YopEcytotoxin Yersinia Jacobietal.,1995

HistoneH2B Yeast Nucleus Flachetal.,1994; Schlenstedtetal.,1995

Tubulin Yeast Microtubules Stearns,1995

Nuf2p Yeast Spindle-polebody Silver,1995;Kahanaetal.,1995

Mitochondrialmatrix Yeast Mitochondria Cox,1995 targetingsignal

Npl3p Yeast Nucleus Corbettetal.,1995

Nucleoplasmin Yeast Nucleus Limetal.,1995

Cap2p Yeast Waddleetal.,1996

Swi6p Yeast Sidorvaetal.,1995




TABLE II continued. PARTIAL LIST OF PROTEInS EXPRESSED AS FUSIOnS TO GFP

Host Cell orProtein Organism Localization References

HMG-R Yeast Hamptonetal.,1996

Bud10p Yeast Halmeetal.,1996

Pmp47 Yeast Peroxisome Dyeretal.,1996

Act1p,Sac6p,Abp1pYeast Cytoskeleton Doyleetal.,1996

p93dis1 Fissionyeast Spindle-polebody& microtubules Nabeshimaetal.,1995

Exu Drosophila Oocytes Wang&Hazelrigg,1994

Mei-S332 Drosophila Centromere Kerrebrocketal.,1995

Tau Drosophila Microtubules Brand,1995

β-galactosidase Drosophila cellmovement Shigaetal.,1996

Streptavidin Insectcells Oker-Blometal.,1996

ObTMVmovement protein Tobacco Filaments Beachy,1995; Heinleinetal.,1995

PotatovirusX coatprotein Potatoplant Viralinfection SantaCruzetal.,1996

GST Zebrafish Cytoplasmic(?) Petersetal.,1995

11β-HSD2 Mammaliancells Endoplasmicreticulum Náray-Fejes-Tóthetal.,1996

MAP4 Mammaliancells Microtubules Olsonetal.,1995

Mitochondrial targetingsignal Mammaliancells Mitochondria Rizzutoetal.,1995

Cyclins Mammaliancells Nucleus,microtubules,orvesicles Pines,1995

PML Mammaliancells Ogawaetal.,1995

Humanglucocor- Mammaliancells Ogawaetal.,1995 ticoidreceptor Careyetal.,1996

Ratglucocorticoid Mammaliancells Rizzutoetal.,1995 receptor Htunetal.,1996

ChromograninB HeLacells Secreted Kaetheretal.,1995

HIVp17protein HeLacells Nucleus Bianetal.,1995

NMDAR1 HEK293cells Membrane Marshalletal.,1995

CENP-B Humancells Nucleus Sullivanetal.,1995



C. GFP Expression in Mammalian Cells Appropriatevectorsmaybetransfectedintomammaliancellsbyavariety

oftechniques,includingthoseusingcalciumphosphate(Chen&Okayama,1988),DEAE-dextran (Rosenthal,1987),various liposome-based trans-fectionreagents(Sambrooketal.,1989),andelectroporation(Ausubeletal.,1994).Anycalciumphosphatetransfectionproceduremaybeused,but we recommend using the CalPhosTM Mammalian Transfection Kit(#K2051-1)forhighcalciumphosphate-mediatedtransfectionefficiencieswithan incubationofonly2–3hours.Likewise,any liposome-mediatedtransfectionproceduremaybeused,butwerecommendusingCLONfectinTM(#8020-1)forhigh,reproducibletransfectionefficienciesinavarietyofcelltypes.Forfurtherinformationoncellculturetechniques,werecommendCultureofAnimalCells,ThirdEdition,R.I.Freshney(1993,Wiley-Liss).

The efficiency of a mammalian transfection procedure is primarilydependentonthehostcellline.Therefore,whenworkingwithacelllineforthefirsttime,itisadvisabletocomparetheefficienciesofseveraltransfec-tionprotocols.ThiscanbestbeaccomplishedusingoneoftheGFPvectorswhichhastheCMVimmediateearlypromoterforhigh-levelexpressioninmostcelllines.

In most cases, GFP expression may be detected by fluorescencemicroscopy,FACSanalysis,orfluorometerassays24–72hourspost-trans-fection,dependingonthehostcelllineused.Ifyouusedelectroporation,waituntil48hourspost-transfectiontoassayorbeginselectiontoallowcells to recover from the electroporation procedure. To visualize GFP-expressingcellsbyfluorescencemicroscopy,growthecellsonasterileglasscoverslipplacedina60-mmcultureplate.Alternatively,aninvertedfluorescencemicroscopemaybeusedfordirectobservationoffluorescentcellsinthecultureplate.

D. GFP Expression in Plants

InArabidopsis,werecommendusingEGFPinsteadofwtGFP,becausethewtcodingsequencecontainsaregionrecognizedinArabidopsisasacrypticplantintron(bases400–483).Thisintronissplicedout,resultinginanonfunctionalprotein(Haseloff&Amos,1995).HaseloffandAmosreportsuccessfulexpressioninArabidopsisofamodifiedversionofGFPinwhichthecrypticintronsequenceshavebeenaltered.ThecrypticintronhasalsobeenremovedbythechangestocodonusageinEGFP,ECFP,andEYFP.A modified GFP gene with the same codon usage as EGFP has beenusedtoexpressGFPinmanyplantsystemsusingavarietyoftransfectiontechniques (Chiuet al., 1996).FunctionalwtGFPhasbeen transientlyexpressedinseveralotherplantspecies,indicatingthatthecrypticintronisunrecognizableinotherplantspecies.


Clontech Laboratories, Inc. www.clontech.com Protocol No. PT2040-1 1� Version No. PR1Y691


E. GFP Expression in YeastwtGFPandGFPuvallexpresswellinyeast.Forexpressioninyeast,theGFPvariantcontainedinClontech’svectorsmustfirstbeisolated(byPCRorbyexcision from theplasmid)and inserted intoanappropriateyeastexpressionvector.Note thatGFPgeneswithhumancodonusage(i.e.,enhancedvariantssuchasEGFP)arenotefficientlyexpressedinyeast(Cormacketal.,1997).Alsonote thatS.cerevisiaestrainscarrying theade2mitochondrialmutationaccumulatearedpigment,whichgivesthecellsanautofluorescencethatinterfereswithdetectionofGFP(Smirnovetal.,1967;Weismanetal.,1987).OurYeastProtocolsHandbook(PT3024-1)containsadditionalinformationonyeastgrowthandmaintenance,preparationofyeastcompetentcells,andtransformationofyeastcells.Toobtainacopy,pleasevisitourwebsiteatwww.clontech.com.Additionally,werecommendGuidetoYeastGeneticsandMolecularBiol-ogy,C.GuthrieandG.R.Finkeds.(Vol.194inMethodsinEnzymology,1991,AcademicPress).




A. Microscopy 1. Conventional microscopy a. Filter sets Althoughyoucanachieveexcellentresultsusingstandardfiltersets,

suchasFITCfilterstodetectEGFP,andrhodamineorpropidiumiodidefilterstodetectDsRed,optimizedfiltersetsfordetectingGFP,GFPvariants,andDsRedhavebeendevelopedbyOmegaOpticalInc.andChromaTechnologyCorp.Ontheirwebsites(www.omega-filters.com;www.chroma.com),youwillfinddetailedinformationabouthow todetectLivingColors®FluorescentProteins.Opticalproperties,recommendeduses,andsampledataareallcarefullydescribedsothatyoucanchoosethefiltersthatbestsuityourneeds.With researchers increasingly interested in multi-color analysis,eachcompanyhasdoneextensivetestingtofindtheoptimalfiltercombinationsforseparatingcyan,green,yellow,andredfluores-cence.Youcanreadaboutthetestresults,includingsuggestionsforexperimentaldesignanddatacollection,andviewcolorimagesbyvisitingeachofthesewebsites.

Ifyoushouldusefluoresceinisothiocyanate(FITC)filters,keepinmindthatthesefiltersetsusuallytransmitexcitationlightat450–500nm—wellabovethemajorexcitationpeak(395nm)ofwtGFP.Thesefilters,therefore,produceafluorescentsignalfromwtGFPthat is several-fold less than would be obtained with filters thattransmitat395nm.However,itisnotadvisabletoexciteat395nmbecauseatthiswavelength,GFPrapidlyphotoisomerizesandlosesthefluorescentsignal.

IncontrasttowtGFP,EGFPhasasingle,strong,red-shiftedexcitationpeakat488nm,makingitwellsuitedfordetectionincommonlyusedequipmentwhichutilizeFITCoptics.Becauselivingcellstoleratelongerwavelengthsbetterduetothelowerenergies,thefluorescentsignalobtainedbyilluminatingEGFPat~488nmismorestableandlesstoxicthanthatofwtGFP.

b. Light source Theemissionintensityfromalightsourcecanvarywithwavelength.

Becausetheintensityoftheexcitationlightdirectlyaffectsthebright-nessofareporter,itisimportanttocomparetheexcitationspectraofyourchosenreporterswiththeemissionspectrumofthelightsource.Conventionalfluorescencemicroscopesareequippedwitheitheramercury-orxenon-arc lamp.Both lampsaresuitable forexcitingfluorescentproteins.Butbeawarethat,whereasthelightintensityfromaxenonlampdeviatesverylittleacrosstheblue,green,yel-low,andredregions,theemissionfromamercurylamphassharppeaksinthecyanandredregions.Therefore,fluorophoresexcited

IV. Detection of GFP, GFP Variants, and DsRed



IV. Detection of GFP, GFP Variants, and DsRed continued

bycyanandredlightmayappearbrighterwhenexcitedbyamercurylamp.Checkthemanufacturers’specificationsfordetailsaboutyourlamp(s).

c. Detection systems Inconventionalmicroscopy,fluorescenceisusuallydetectedwith

eitheraphotographic(35-mm)ordigitalcamera(whichfrequentlyharborsacooledcharge-coupleddevice,aCCD).Oneadvantageofa35-mmcameraisthatitcanrecordthe“true”color(asseenbyeyethroughthemicroscope)ofasinglefluorescentproteinaswellasthemixedcolorofcolocalizedfluorescentproteins.Butrecordinganimagewitha35-mmcameramayrequirealonger-than-averageexposuretime,effectivelyphotobleachingthesample.Digitalcameras,ontheotherhand,areusuallymoresensitivethan35-mmcamerasbut(unlessyouuseacolordigitalcamera)requireimageanalysissoftwaretoproduce“pseudo-colored”data.

d. Multicolor analysis Withthedevelopmentofoptimizedfiltersetsandtheintroduction

ofourredfluorescentprotein,DsRed,it’snowpossibletoseparateasmanyasthreefluorescentreporters(cyan,yellow,andred)bymicroscopy. Some investigators have even distinguished all fourcolors(cyan,green,yellow,andred)usingflowcytometry(Hawleyetal.,2001).Infact,manyexamplesofmulticoloranalysiscannowbefoundintheliterature(afewofwhicharelistedinTableIII),andwerecommendyousurveytheliteratureforhelpindecidingwhichcombinationwillworkbestforyou.Here,weofferthefollowingrec-ommendationsfortwo-coloranalysis:

• Fordual-labelingexperiments,werecommendusingDsRed2withEGFP.OneadvantageofusingDsRed2withEGFPisthatanoverlayoftheproteins’emissionsappearsyellow,soyoucandistinguishbetweentheindividualproteinsandtheoverlap.

• ECFPandEYFPcanalsobeusedtogethertocarryoutdouble-labelingexperiments(Greenetal.,2000;Ellenbergetal.,1999;July1999Clontechniques).WetestedtheopticalseparationofECFPandEYFPwithconventionalfluorescencemicroscopy.Inonestudy,weexaminedHeLacellsexpressingECFPandEYFP,targetedtothemitochondriaandnucleus,respectively(Greenetal.,2000).Tovisualizethefluorescence,weusedaZeissAxioskopequippedwitha100Wmercuryarclampandfilter sets for detecting ECFP (Omega Optical’s XF114) andEYFP(OmegaOptical’sXF104). Imageswerecapturedwitha35-mmcamera.Using thissetup, the twocolorsarewellseparated; no bleed-through was evident. During the initialexposure,(i.e.,duringthefirst~15sec),theseproteinsdisplay




similarintensities;however,becauseECFPphotobleachesfaster than EYFP, unequal intensities become obvious afterseveralmoresecondsofillumination.

TABLE III. MULTI-COLOR AnALYSIS wITH LIVInG COLORS® PROTEInS

Type of Analysis Application Recommended References* Combination

Two-color Microscopy DsRed+EGFP(GFP) October1999Clontechniques Handler&Harrell,2001

ECFP+EYFP Greenetal.,2000 Stuurmanetal.,2000 October1999Clontechniques

Three-color Microscopy DsRed+ECFP+EYFP Bloembergetal.,2000 October1999Clontechniques

Four-color Flowcytometry DsRed+EGFP+EYFP Hawleyetal.,2001 +ECFP July2001Clontechniques

*Also,seePattersonetal.,(2001).

2. Laser-scanning microscopy Ifyouareusingalaser-scanningconfocalfluorescencemicroscope,

yourexcitationsourcewillusuallybeanargon-ionorkrypton-ionlaser,orboth.Withthe488-nmlineoftheargonlaser,youcanexciteEGFP,EYFP,andDsRed.Butyou’llneedthe458-nmlinefromanargonlaseror413-nmlinefromakryptonlasertoproducestrongemissionsfromECFP(Hawleyetal.,2001;Ellenbergetal.,1999).Thekrypton-ionlasermayalsobeusedtoexciteDsRed; its568-nmline isclosetoDsRed’s excitation maximum (Hawley et al., 2001). With so manylaser configurations now available, we recommend you consult themanufacturer(s)toobtainmoredetailedinformationaboutaparticularsetup.Comparethelaserspecificationswiththeexcitationspectraofthefluorescentproteinsyouplantouse.

3. Multi-photon laser-scanning microscopy Multi-photonexcitationmicroscopyhaswontheapprovalofmanyre-

searcherswhoseekotheralternativesforresolvingfluorescentlylabeledproteinsinvivo.Itsadvantageshavebeenwidelyreported(Piston,D.W.,1999;Potter,S.M.,1996;Marchant,J.S.,2001).Chiefamongthemis theability toexcitefluorophoreswith low-energy infrared light. IRlightnotonlypenetratestissuemoreeffectivelythanvisiblelight,italsominimizesphotobleachingandcauseslessphotodamage.Inprinciple,allLivingColorsFluorescentProteins,includingDsRed(Jakobsetal.,2000),aresuitableforuseinmulti-photonapplications.




4. Fluorescence Microscopy Theprotocolprovidedbelowisonepossiblemicroscopyprocedure.

Otherequallysuitableandmoredetailedmicroscopyproceduresmaybefoundelsewhere(e.g.,Ausubeletal.,1995etseq.)

Materialsrequired: • 70%Ethanol • Dulbecco’sPhosphatebufferedsaline(DPBS;pH7.4) • DPBS/4%paraformaldehyde(pH7.4–7.6)

Add4gofparaformaldehydeto80mlofDPBS.Heattodissolve.Oncethesolutionhascooled,readjustthepHifnecessary,thendilutetoafinalvolumeof100ml.Storeat–20°C.

• Rubbercement,moltenagarose,orcommercialmountingmedium(e.g.,ProLong®AntifadeKit,MolecularProbes)

Fluorescencemicroscopyprocedure Inatissueculturehood: a.Sterilizeaglasscoverslipwith70%ethanol. b.Placethecoverslipinasteriletissueculturedish. c.Plateand transfect cells in the tissue-culturedish containing the

coverslip. note: Somecelltypesmaynotadheretotheglasscoverslip.Inthesecases,you

mayneedtopre-treattheglasscoverslipwithasubstratethatpromotescelladhesion(e.g.,lamin,orpoly-d-lysine,orboth).Asanalternativetoglasscoverslips,youmighttryculturingcellsdirectlyonBDFalconTMCultureSlides.

c.At theendofthecultureperiod,removethetissueculturemediaandwashoncewithDPBS.

d.Fixingcells i.AftercellshavebeenwashedwithDPBS,add freshlymade

DPBS/4%paraformaldehydedirectlytothecoverslip. ii.Incubatecellsinsolutionatroomtemperaturefor30min. iii.WashcellstwicewithDPBS.AllowcellstosoakinDPBSfor

10minduringeachwash. e.Mountingthecoverslipontoaglassmicroscopeslide i.Carefully remove the coverslip from the plate with forceps.

Paycloseattentiontowhichsideofthecoverslipcontainsthecells.

ii.Placeatinydropofcommercialmountingsolution(e.g., Pro-Long®AntifadeKit,MolecularProbes)ontheslide,andallowthecoversliptoslowlycontactthesolutionandtoliedownontheslide,cellsidedown.



iii.Carefully aspirate the excess solution around the edge ofthecoverslipusingaPasteurpipetteconnectedtoavacuumpump.

iv.Ifdesired,sealthecoversliptothemicroscopeslideusingmoltenagarose,rubbercement,orblacknailpolish.

v.Allowtodry.Thedryingtimemayvarydependingonthemount-ingsolutionused.

vi.Examine slides by fluorescence microscopy. Once fixed,cellscanbestoredinthedarkat4°C.

B. Flow Cytometry 1. Multicolor analysis and cell sorting SeveralinvestigatorshavesortedGFP-expressingcellsbyflowcytometry

(Cheng&Kain,1995;Roppetal.,1995;Yu&vandenEngh,1995).FACSanalyseshavebeencarriedoutwithplantprotoplasts,yeast,E.coli,andmammaliancellsexpressingGFP(Atkins&Izant,1995;Feyetal.,1995;Sheenetal.,1995;Chengetal.,1996).MaximalemissionisachievedwhenEGFPisexcitedat488nm,whichcorrespondsperfectlytothe488-nmlineofargon-ionlasersusedinmanyflowcytometers.Infact,thisvariantwasselectedspecificallyonthebasisofitsincreasedfluorescenceinflowcytometryassays.

SinceEGFP,EYFP,andDsRedcanallbeexcitedbyasingle laserexcitationwavelength(488nm),two-color(EGFP/EYFP;Lybargeretal.,1998)andthree-color(Hawleyetal.,2001)flowcytometricanalysesarepossible.ECFPandEYFParenot recommended fordual-coloranalysiswithflowcytometrybecausetheargonlaser,usedforexcita-tioninmostflowcytometers,doesnotefficientlyexciteECFP.ToexciteECFP,yourflowcytometermustbeequippedwithakryptonlasertunedto413nm(Ellenbergeretal.,1998&1999)oranargonlasertunedto458nm(Hawleyetal.,2001).

EGFPandDsRedareperhapsthebestcombinationforcellsortingandtwo-coloranalysisbyflowcytometry.Theseproteinscanbeco-excitedbythe488nmlineoftheargonlaserandtheiremissionscanbecon-venientlyrecordedthroughtheFL-1(green)andFL-2(red)channels.Theanalysiscanbefurtherrefinedbyusingoptimizedfilterssetsandbyadjustingthecompensationsettings.





2. Detecting fluorescent proteins in ethanol-treated cells Whenexpressedasaqueoussolubleproducts,fluorescentproteins

mayleakfromethanol-treatedcells;ethanolpermeabilizestheplasmamembrane.Toavoidthisproblem,wesuggestyouuseparaformalde-hydeifyouplantofixfluorescentprotein-expressingcells.Otherwise,ifyouwishtomeasuretransfectionefficiency,butplantofixcellswithethanol,werecommendusingEGFP-F,ECFP-Mem,orEYFP-Mem,describedbelow.

Insomecases,youmaynoticeagradualincreaseinbackgroundfluo-rescence inethanol-treatedcellsduringflowcytometry.ArinsewithPBSsupplementedwith1%BSAafterethanolfixation,orlongfixationtimes(18–24hr),eliminatesthisproblem(D.Ucker,pers.comm.).

a.EGFP-Fispost-translationallyfarnesylated,soitremainsattachedtotheinnerfaceoftheplasmamembraneevenduringtreatmentwithethanol.EGFP-Fcontainsashort,C-terminalfarnesylationsignalfromc-Ha-Ras(Aronheimetal.,1994;Hancocketal.1991).ThesignaltargetsEGFP-FtotheplasmamembranewithoutalteringtheintrinsicfluorescentpropertiesoftheEGFPchromophore(Jiang&Hunter,1998).

b.ECFP-Mem&EYFP-MemareN-terminalfusionsthatcontaintheN-terminal20aminoacidsofneuromodulin(GAP-43;Moriyoshietal.,1996).Duringpost-translationalprocessing,apalmitoylgroupisaddedtotheneuromodulindomainofthefusionprotein.Themodi-ficationtargetsthefluorescentproteinprimarilytotheinnerfaceoftheplasmamembrane.Asmallamountofthefusiondoesbindothercellularmembranes.




C. Quantitative Fluorometric AssayRecombinantGFP(orEGFP)canbeusedinfluorometricassaystoconfirmandmeasureGFPexpression.Aftergeneratingastandardcurveusingknownamountsofrecombinantprotein,comparethefluorescenceintensitiesofyourexperimentalsamplestothestandardcurvetodeterminetheamountofGFPexpressedinthesample.AdetailedmethodforquantitationofwtGFPlevelsusingtheTD-700fluorometerisavailablefromTurnerDesigns,Inc(www.turnerdesigns.com).

Whengeneratingthestandardcurve,therecombinantproteinshouldbedilutedinthesamebufferastheexperimentalsamples.Insomeexpressionsystems,GFPcanbedetecteddirectlyinthefluorometer(simplysuspendcellsinanisotonicbuffersuchasPBS);inothercases,cellswillneedtobedisruptedinordertooptimallydetectGFPfluorescence.

Figure6showsacomparisonofthefluorescenceintensityofGFPdetectedinsonicationbufferand inaclearedbacterial cell lysate.These resultsillustratethatthesensitivityandthelinearrangeofdetectionofGFParerelativelyunaffectedbythecelllysatebackground.

Figure 6. Comparison of fluorescence intensity of GFP in sonication buffer versus GFP in cell lysates.LBwasinoculatedwithacultureofuntransformedJM109cellsandincubatedovernightat30°C.Celllysateswerepreparedasdescribedintheprotocol.SerialdilutionsofrGFPwerepreparedinbothsonicationbufferandJM109celllysate.FluorescenceintensitywasmeasuredinamodifiedHoeferPharmaciaBiotech,Inc.DyNAQuant200Fluorometerusinganexcitationfilterof365nmandanemissionfilterof510nm.(RFU=relativefluorescenceunits)

100

1

2

�

4

2

Log GFP (ng)

GFP in 6.2� mg JM109 lysate

GFP in sonication buffer

Log

RFU

� 4




ThefollowingprocedureshavebeendevelopedusingourRecombinantGFPProtein(rGFP#8360-2).Useanexcitationfilterof365nmandanemissionfilterof510nmwhenassayingwtGFPorGFPuv,andanexcitationfilterof450–490nmandanemissionfilterof510nmwhenassayingEGFP.

1.Materialsrequired • Sonicationbuffer(pH8.0) 50 mM NaH2PO4 10 mM Tris-HCl(pH8.0) 200 mM NaCl AdjustpHto8.0.Storeatroomtemperature. • PBS(pH7.4) 2.GeneratingaGFPstandardcurve: a.PrepareserialdilutionsofrGFPinsonicationbuffer,deionizedH2O,

or10mMTris-HCl(pH8.0). note: Samplescanbestoredat–20°C. b.Assaydilutionsinafluorometer. c.Plotthelogoftherelativefluorescenceunits(RFU)asafunctionof

thelogoffold-dilutionofeachsamplesuchasthestandardcurveshowninFigure7.

3.Measuringfluorescenceintensityofbacterialsamples: a.Pellet20mlofbacterialcellculture. b.Removethesupernatantandfreezepelletat–70°C. c.Washpelletoncewith1.6mlofPBS. d.Resuspendpelletin1.6mlofsonicationbuffer. e.Prepare2-foldserialdilutionsinsonicationbuffer. f.Assaydilutionsinafluorometer. g.Compareresultswiththoseofthestandardcurvetodeterminethe

amountofGFPproteinexpressedintheexperimentalsamples. 4.Preparationofbacterialcelllysates: a.Pellet100mlofbacterialcellcultureandremovethesupernatant. note:Cellpelletsmaybestoredat–70°Cforlateruse. b.Washpellettwicewith4mlofPBS. c.Resuspendpelletin4mlofsonicationbuffer. d.Freeze/thawsampleinadryice/ethanolbathfivetimes. e.Vortexsamplefor1min,thenincubateonicefor1min. f.RepeatStepetwomoretimes. g.Runsamplethroughan18-gaugeneedleseveraltimes.



Figure 7. Relative fluorescence intensity of two-fold serial dilutions of a suspension of GFP-transformed cells in sonication buffer. LBwasinoculatedwithJM109cellstransformedwithaplasmidencodingwtGFPandincubatedovernightat30°C.Fluorescenceintensitywasmeasuredbyreading5-µlsamplesof2-folddilutionsinamodifiedHoeferPharmaciaBiotech,Inc.DyNAQuant200Fluorometerusinganexcitationfilterof365nmandanemissionfilterof510nm.

00

1

2

1

Log Fold Dilution

DR60�14pr.bw/GFP.fi.sb

Log

RFU

2

h.Transfercelllysatetoa1.5-mlmicrocentrifugetubeandcentrifugeat12,000rpmfor5minat4°C.

i.Collectthesupernatantanddeterminetheproteinconcentrationus-ingastandardassay(e.g.,Bradford,Lowry,etc.;Scopes,1987).

j.Assaythedilutionsinafluorometer. k.Comparetheresultswiththestandardcurvetodeterminetheamount

ofGFPexpressedintheexperimentalsamples.


Clontech Laboratories, Inc. www.clontech.com Protocol No. PT2040-1 2� Version No. PR1Y691


A. General InformationOurRecombinantGreenFluorescentProtein(rGFP;#8360-2),RecombinantEGFP(rEGFP;#8365-1),andRecombinantGFPuv(rGFPuv;#8366-1)arepurifiedfromtransformedE.coliusingamethodwhichensuresoptimalpurityoftherecombinantproteinandmaintenanceofGFPfluorescence.Allproteinsare27-kDamonomerswith239aminoacids.TheexcitationandfluorescenceemissionspectrafortherGFPisidenticaltoGFPpurifiedfromAequoreavictoria(Chalfieetal.,1994).Thepurifiedproteinsretaintheirfluorescencecapabilityundermanyharshconditions (Section II.F)andaresuitableascontrolreagentsforGFPexpressionstudiesusingtheLivingColorslineofGFPreportervectors.Applications of purified rGFP and rGFP variant proteins include useas standards for SDS or two-dimensional PAGE, isoelectric focusing,Westernblotanalysis,calibrationoffluorometersandFACSmachines,fluo-rescencemicroscopy,andmicroinjectionofGFPintocellsandtissues.

B. rGFP and rGFP Variants as Standards in western BlotsWhenusedasastandardforWesternblottingapplicationsinconjunctionwiththeLivingColorsA.v.PeptideAntibody(#8367-1,-2),therecombinantproteinscanbeusedtocorrelateGFPexpressionlevelstofluorescenceintensityortodifferentiateproblemswithdetectionofGFPfluorescencefromexpressionofGFPprotein.Useastandardprocedurefordiscontinuouspolyacrylamidegelelectropho-resis(PAGE)toresolvetheproteinsonaone-dimensionalgel(Laemmli,1970).Ifanotherelectrophoresissystemisemployed,thesampleprepara-tionshouldbemodifiedaccordingly.Justpriortouse,allowrGFPproteintothawatroomtemperature,mixgentlyuntilsolution isclear,andthenplacetubeonice.Onaminigelapparatus,25–75µgof lysateproteinper lane is typicallyneededforsatisfactoryseparation(i.e.,discretebandingthroughout themolecularweightrange)ofaproteinmixturederivedfromawholecellortissuehomogenate.IfrGFPistobeusedasaninternalstandardinaCoo-massieblue-stainedminigel,werecommendloading500ngofrGFPperlane.IfrGFPisaddedtoatotalcell/tissuelysateorothercrudesample,theamountoftotalproteinloadedperlanemustbeoptimizedfortheparticularapplication.ForWesternblottingapplications,werecommendloading40–400pgofrGFP(orrGFPvariant)perlaneforastrongpositivesignal.Generally,rGFPwillnotfluoresceonanSDSgeloraWesternblot.

V. Purified Recombinant GFP and GFP Variants



C. rGFP and rGFP Variants as a Control for Fluorescence MicroscopyThefollowingtwoprotocolsareforuseofrGFPorrGFPvariantsasacon-trolonmicroscopeslidesinfluorescencemicroscopy.ThepurifiedproteinsmaybeusedtooptimizelampandfiltersetconditionsfordetectionofGFPfluorescence,orasaqualitativemeanstocorrelateGFPfluorescencewiththeamountofproteinintransfectedcells.

1. Unfixed samples Usethismethodforlivecellfluorescenceorothercaseswhereafixa-

tionstepisnotdesired. a.Perform1:10serialdilutionsofthe1.0mg/mlrGFPorrGFPvariant

stocksolutionwith10mMTris-HCl(pH8.0)toyieldconcentrationsof0.1mg/mland0.01mg/ml.

notes: • Thesedilutionsshouldsufficeasapositivecontrol.The1.0mg/mlsolutionwill

giveaverybrightfluorescentsignalbymicroscopy. • Thediluted samples canbealiquotedandstored frozenat –70°C for up to

1yrwithnolossoffluorescenceintensity.Avoidrepeatedfreeze-thawcycleswiththesamealiquot.

b.Using a micropipette, spot 1–2 µl of diluted protein onto themicroscopeslide.Ifusingaslidethatcontainsamountedcoverslip,positionthespotseveralmillimetersawayfromthesamplesuchthatasecondcoverslipcanbeaddedovertheproteinspot.

c.Allow the protein to air-dry for a few seconds, and mark thepositionofthespotontheothersideoftheslidetoaidinfocusing.

d.Add a coverslip over the spot using a 90% glycerol solution in100mMTris-HCl(pH7.5).

e.Fluorescencefromthespotisbestviewedatlowmagnification,usingeithera10Xor20Xobjectivelens.

2. Fixed samples Insomecasesitmaybenecessarytofixtherecombinantproteinto

themicroscopeslidepriortomicroscopy.Thiscanbedonebydippingthesectionofthemicroscopeslidecontainingtheair-driedproteinspot(afterStep1.cabove)into100%methanolfor1min.AllowtheslidetodrycompletelyandplaceacoverslipoverthesampleasinStep1.dabove.

V. Purified Recombinant GFP and GFP Variants continued

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT2040-1 �0 Version No. PR1Y691


A. Applications for GFP Antibodies WeofferthreeGFP-specificantibodies:

• Living Colors®A.v. PeptideAntibody (#8367-1, -2) detects all GFPvariants.TheA.v.PeptideAntibody,whichisalsoavailableconjugatedtohorseradishperoxidase(HRP),isrecommendedforWesternanaly-sis.

• LivingColors®A.v.(JL-8)MonoclonalAntibody(#8371-1,-2),amousemonoclonal(subclassIgG2a),recognizesallLivingColorsGFPvari-ants,includingourenhancedanddestabilizedproteins,fusionstotheseproteins,andpurifiedrecombinantGFP.TheJL-8MonoclonalisidealforWesternandELISAapplications.

• LivingColors®Full-LengthA.v.PolyclonalAntibody(#8372-1,-2)wasspecifically developed and tested for immunoprecipitating GFP andGFPvariants. ItalsodetectsdestabilizedvariantsandfusionstoallLivingColorsGFPproteins.

WealsoofferDsRed-specificantibodies: • Living Colors® D.s. Peptide Antibody (#8370-1, -2) recognizes wt

DsRedandDsRedvariants suchasDsRed1-E5andDsRed2.TheantibodyalsobindsDsRedfusionswhentheproteinofinterestisfusedtotheN-terminusofDsRedoroneof itsvariants.BecausetheD.s.PeptideepitopeoftenbecomeshiddenwhenproteinsarefusedtotheC-terminus of DsRed, you may have difficulty detecting C-terminalfusionswiththisantibody.Raisedinrabbitsandaffinity-purified,thispolyclonalantibodyisrecommendedforuseinWesternblottingandimmunoprecipitation.TheD.s.PeptideAntibodydoesnotcross-reactwithanyAequoreavictoriaGFPvariants.

• LivingColors®DsRedMonoclonalAntibody(#8374-1,-2)recognizesdenaturedformsofwild-typeDsRedanditsvariants,includingDsRed1,DsRed2,andDsRed1-E5.RecommendedforWesternblottingapplica-tionsonly,thisantibody(mouseIgG)bindsDsRed1andDsRed2evenwhen theproteinsareexpressedas fusions tootherproteins.BothN-andC-terminalfusionsarerecognized.

VI. GFP Antibodies

Protocol No. PT2040-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR1Y691 �1


VI. GFP Antibodies continued

B. Immunoprecipitation with the Living Colors A.v. Peptide Antibody 1. Solutions Required • PBS • Lysisbuffer PBScontaining0.5%TritonX-100,5mMEDTA,andthefollow-

ingproteaseinhibitors(allavailablefromSigma):PMSF,0.1mM;pepstatinA,10µM;leupeptin,10µM;aprotinin,25µg/ml.

• LivingColorsPeptideAntibody • ProteinAorProteinGagarosebeads(PierceorBioRad) Use20–30µlofbeadspersample.Spindownbeadstoremove

theethanol,thenwashbeadstwiceinPBS.Aspiratethesuperna-tantfromthepelletofbeads.

2. Procedure a.Spindown~1x106cells(orcellsfromone100-mmdish)andwash

twiceinPBS.Resuspendin1.2mloflysisbuffer. b. Incubateat4°Cfor30minwithcontinuousgentleinversion. c. Clearthelysatebycentrifuginginamicrocentrifugeat12,000rpm

for20minat4°C. d. Transfer1mlofsupernatanttoa1.5-mlmicrocentrifugetube. e. Add5µgofLivingColorsA.v.PeptideAntibodytothesupernatant.

Incubateat4°Cfor2–3hrwithcontinuousgentleinversion. f. Addtheantibody/lysatemixturetothepelletofProteinA(orProtein

G)agarosebeads. g. Vortexandincubateat4°Cfor2hrwithcontinuousgentleinver-

sion. h. Washthebeads5timeswithPBS.Foreachwash,add1mlofPBS

andincubatefor2minwithcontinuousgentleinversion.Aspiratethesupernatantfromthepelletofbeads.

3. Sample analysis: a. Washthebeadswith1mlofLysisBuffer. b. Repeatthiswashprocedurefourtimes. c. Add30µlof2XSDS-PAGEsamplebuffertobeads. d. Vortex,thenboilfor2min. e. Repeatstepd. f. Centrifuge inamicrocentrifugeat12,000rpmfor1minat room

temperature. g. Electrophorese10–20µlofsupernatantonanSDS-polyacrylamide

gel(a12%gelwillresolveGFPat27kDa).AnalyzetheresolvedproteinsonaWesternblot,bysilverorCoomassiebluestaining,orbyautoradiography.



VII. Troubleshooting Guide

A. Potential Difficulties in Using GFP Fluorescence • VariabilityintheintensityofGFPfluorescencehasbeennoted.Thismay

bedueinparttotherelativelyslowformationoftheGFPchromophoreandtherequirementformolecularoxygen(Heimetal.,1994).

• The slow rate of chromophore formation and the apparent stabilityofwtGFPmayprecludetheuseofGFPasareportertomonitorfastchangesinpromoteractivity(Heimetal.,1994,Davis,I.etal.,1995).ThislimitationisreducedbyuseofEGFPanddestabilizedGFPs,whichacquirefluorescencefasterthanwtGFP(Heimetal.,1994).

• ThewtGFPcodingsequencescontainacrypticplantintron(betweennucleotides400and483)thatisefficientlysplicedoutinArabidopsisandresultsinanonfunctionalprotein(Haseloff&Amos,1995).FunctionalwtGFPhasbeentransientlyexpressedinseveralotherplantspeciesindicatingthatthecrypticintronmaynotberecognizedorrecognizedlessefficientlyinthesespecies.EGFP,ECFP,andEYFPdonotcontainthecrypticintronandcanbeusedforexpressioninplants.

• SomepeoplehaveputGFPexpressionconstructsintotheirsystemandfailedtodetectfluorescence.Therecanbenumerousreasonsforfailure,includinguseofaninappropriatefilterset,expressionofGFPbelowthelimitofdetection,andfailureofGFPtoformthechromophore.RecombinantGFPProteinandaGFP-specificantibody,suchastheLivingColorsA.v.PeptideAntibody,maybeusedtotroubleshootGFPexpressioninthesecases.SinceGFPuvandEGFPfluorescebrighterthanwtGFP,theyarebetteralternativesforexpressionofGFP.

• GFPtargetedtoalowpHenvironmentmaylosefluorescence. • Ethanol-fixed cells will lose fluorescence because the soluble fluo-

rescentproteinleaksthroughthepermeabilizedplasmamembranes.Use pEGFP-F, pECFP-Mem, or pEYFP-Mem Vectors to avoid thisproblem.

B. Requirements for GFP Chromophore Formation • FormationofthechromophoreinwtGFPappearstobetemperature

sensitive;however,themutationsinEGFP,EYFP,ECFP,andGFPuvsuppressthisthermosensitivity.Insomecases,E.coli,yeast,andmam-maliancellsexpressingwtGFPhaveshownstrongerfluorescencewhengrownatlowertemperatures(Heimetal.,1994;Ward,pers.comm.;Limetal.,1995;Pines,1995;Ogawaetal.,1995).Hence,incubationatalowertemperaturemayincreasethefluorescencesignalobtainedwhenusingwtGFP.

• GFPchromophoreformationrequiresmolecularoxygen(Heimetal.,1994;Davis,D.F.etal.,1995);therefore,cellsmustbegrownunderaerobicconditions.

Protocol No. PT2040-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR1Y691 ��


VII. Troubleshooting Guide continued

C. Photobleaching or Photodestruction of Chromophore • Exciteat470nmforwtGFP,360–400nmforGFPuv,and488nmfor

thered-shiftedGFPexcitationvariants.Excitationatthe395-nmpeakforwtGFPmayresultinrapidlossofsignal.ForGFPuv,usethelongestpossibleexcitationwavelength tominimize the rateofphotobleach-ing.

•Atungsten-QTHorargonlightsourceispreferable.MercuryandxenonlampsproducesignificantUVradiationwhichwillrapidlydestroythechromophoreunlessstronglyblockedbyappropriatefilters.

D. Autofluorescence • Somesamplesmayhaveasignificantbackgroundautofluorescence,

e.g.,wormguts(Chalfieetal.,1994;Niswenderetal.,1995).Aband-passemissionfiltermaymaketheautofluorescenceappearthesamecolorasGFP;usingalong-passemissionfiltermayallowthecoloroftheGFPandautofluorescencetobedistinguished.UseofDAPIfiltersmay also allow autofluorescence to be distinguished (Brand, 1995;Pines,1995).

• Mostautofluorescenceinmammaliancellsisduetoflavincoenzymes(FAD and FMN;Aubin, 1979) which have absorption and emissionmaximaat450and515nmrespectively.ThesevaluesareverysimilartothoseforwtGFPandthered-shiftedGFPvariants,soautofluores-cencemayobscuretheGFPsignal.TheuseofDAPIfilterswhenusing450/515nmlightforexcitationmaymakeautofluorescenceappearbluewhiletheGFPsignalremainsgreen.Inaddition,somegrowthmediacancauseautofluorescence.Whenpossible,performmicroscopyinaclearbuffersuchasPBS,ormediumlackingphenolred.Also,whenselecting forGFPexpression inmammaliancells,youmaywant toexcludethehighestexpressingclones,whichwillexhibitmoreback-groundfluorescenceinculture(D.Piston,pers.comm.).

• Some cell types can produce a speckled autofluorescence patternwhichislikelyduetomitochondriallyboundNADH(Aubin,1979).Thisproblemisminimizedwithexcitatorylightaround488nm,asopposedtoUVexcitation(Niswenderetal.,1995).Therefore,werecommendusingEGFPforexpressioninthesesystems.

• Formammaliancells,autofluorescencecanincreasewithtimeincul-ture.Forexample,whenCHOorSCIcellswereremovedfromfrozenstocksandreintroducedintoculture,theobservedautofluorescence(emissionat520nm)increasedwithtimeuntilaplateauwasreachedaround48hours(Aubin,1979).Therefore,insomecasesitmaybepreferabletoworkwithfreshlyplatedcells.




• Alwaysuseamock-transfectedcontroltogaugetheextentofautofluo-rescence.

• For fixed cells, autofluorescence can be reduced by washing with0.1%sodiumborohydrideinPBSfor30minafterfixation.

E. Considerations for Mammalian Expression • Useanenhancedfluorescentvariant(ECFP,EGFP,orEYFP)instead

ofwtGFP.Thebrighterfluorescenceandimprovedtranslationofthesevariantsmakesthemeasiertodetect.

•VerifyyourGFPvariantplasmidconstructandconcentrationwitharestrictiondigest.Verifythatallsubcloningstepshavebeendonecor-rectly,keepinginmindspecificrestrictionsitesinsomevectorswhichareinactivatedbymethylation.OurEGFPSequencingPrimersmaybeusedtoverifysequencejunctions.

• Besurethevectoriscompatiblewithyourcelltype.FindoutiftheCMVpromoterhasbeenusedpreviously for transientexpression inyourcells.

•Useanotherassaytoestimatetransfectionefficiency.Transfectwithasecondreporterplasmidthatcontainsthesamepromoterelement.Forexample,usepEGFPLuc(#6169-1)orpHygEGFP(#6014-1).WithpEGFPLuc (#6169-1), you can determine transfection efficiency byfluorescencemicroscopyandobtainquantitativedatausingastandardluciferaseassay.

•ExpressionofGFPproteincanbeverifiedbyWesternanalysisusingeithertheLivingColorA.v.PeptideAntibody(#8367-1,-2)ortheLivingColorsFull-LengthA.v.PolyclonalAntibody(#8373-1,-2).

F. Optimizing Microscope/FACS Applications • Ingeneral,optimalvisualdetectionofGFPfluorescencebymicroscopy

isachievedinadarkenedroom,afteryoureyeshaveadjusted. •ChoosethefiltersetthatisoptimalfortheGFPvariantthatyouareus-

ing.Ingeneral,conditionsusedforfluoresceinshouldgivesomesignalwithallvariantsexceptGFPuv.Autofluorescencemaybeaprobleminsomecelltypesororganisms.FormoreinformationonthefiltersetsrecommendedforGFPanditsvariants,seeSectionIV.

•Tocheckthemicroscopesetup,spotasmallvolumeofpurifiedrecom-binantGFPprotein(e.g.,rGFP,rEGFP,orrGFPuv)onamicroscopeslide.Asacrudesubstitute,anysourceoffluoresceincanbeused,suchasafluorescein-conjugatedantibodyoravidin-fluorescein.

• GFPfluorescenceisverysensitivetosomenailpolishesusedtosealcoverslips(Chalfieetal.,1994;Wang&Hazelrigg,1994).Inplaceofnailpolishformountingcoverslips,werecommendmoltenagarose,rubbercement,orcommercialmountingsolution.




• ExcitingGFPintenselyforextendedperiodsmaygeneratefreeradicalsthataretoxictothecell.Thisproblemcanbeminimizedbyexcitationat450–490nm.

G. Anomalous Band in some GFP Plasmid Preparations Wehaveoccasionallyobservedabandrunningatapproximately500bp

insome(butnotall)plasmidpreparationsoftheGFPvectorsthathavethebackbonecarryingthekanamycinresistancegeneandthef1origin(i.e.,pEGFP-1,pEGFP-N1,-N2,-N3,-C1,-C2,-C3,pEYFP-C1,-N1,pEYFP-1,andpECFP-N1,-C1,and-1).ThepresenceandlevelofthisbandvariesgreatlyfromoneplasmidDNApreparationtoanother.Wehavenotthoroughlycharacterizedthisband;however,itappearstobeasmallcircularDNA,possiblysingle-stranded,generatedasabyproductofplasmidreplication.Asfaraswecanascertain,thisbanddoesnotinterferewithligatinginsertsintothevectors,doesnotaffecttransformationortransfectionefficiencies,anddoesnothaveanydeleteriouseffectonmammaliancells.However,ifyouwishtoavoidthepresenceofthisbandinyourplasmidpreps,tryanynon-alkalinebacteriallysisprocedure(Sayersetal.,1996)orgel-purifythemajorbandafteralkalinelysis.



VIII. References

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Protocol No. PT2040-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR1Y691 �9



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