MODERN METHODS in BIOCHEMISTRY - unifr.ch · modern methods in biochemistry •protein modification •protein crosslinking •protein staining •antibody modification •immunoprecipitation

MODERN METHODS inBIOCHEMISTRY

•PROTEIN MODIFICATION•PROTEIN CROSSLINKING•PROTEIN STAINING•ANTIBODY MODIFICATION•IMMUNOPRECIPITATION•METABOLIC LABELLING

Affinity chromatography :

• matrices – preparation• Coupling of Ligand• detection of reactive groups

Affinity chromatography :Principle of method

Mouse brain

Solubilise & homogenise

50,000 x g

supernatant

Affinity Chromatography Immunoprecipitation

eluted complexes

Gel filtration

SDS-PAGE

Affinity chromatography Example

Affinitychromatographymatrice preparation& introduce spacer

Affinity chromatography :Coupling of Ligand

Affinity chromatography Example

COMMONLY USED AGENTS1. Cyanogen bromide

• Cyanogen bromide (CNBr) is one of the most widelyused linkers in affinity chromatography. It reacts withhydroxyl groups in agarose and other polysaccharidematerices to produce a reactive linker. The linker canbe connected to ligands or spacers which containprimary amine groups (such as proteins).

• Cyanogen bromide is extremely toxic as it releaseshydrogen cyanide on acidification.

COMMONLY USED AGENTS2. Bis-Expoxides (Bis-oxirane)• Bis epoxides react readily with both

hydroxyl or amino-containing gels atalkaline pH.

• This produces a long chain hydrophilicreactive oxirane. This can then belinked to ligands.

• The long chain oxirane itself acts as aspacer.

COMMONLY USED AGENTS3. Carbonyldiimidazole

• N,N'carbonyldiimidazole (CDI) is reacts withpolysaccharides to form imidazole carbonatederivatives. These derivatives then react with ligandscontaining primary amino groups at an alkine pH togive stable carbonate derivatives.

4. Sulphonyl chloride• Reacts with hydroxyl groups on the matrix to

form sulphonyl esters. These esters react with aminoand thiol groups of the ligand.

5. Sodium periodate (NaIO4)• Reacts with diol groups on polysaccharide matrices to

form reactive aldehydes. These react with primary aminesto form Schiff's bases.The Schiff's bases can be stabilizedby reduction with sodium borohydride.

• Periodate is easy to use and non-toxic. The product isstable.

6. Glutaraldehyde• Activates amino or amide groups on polyacrylamide

matrices or on agarose with amine spacers. These groupscan then react with primary amine groups on the ligand.

Tags and Fusion sequences• Many vectors are now engineered with DNA sequences encoding a specific

peptide (purification tag) that, when fused to the expressed protein, may beused for one-step purification of the recombinant protein by high-affinitybinding. That is molecular biologists design expression systems such that therecombinant protein can be easily purified using affinity chromatography.

• The technique involves linking the gene coding for tag to that codingfor the protein of choice. When the latter is expressed, the resulting tagis also produced and is linked to the desired protein. The tag isselected such that it can be readily be bound a particular affinitychromatography material.

• Example 1 : 6xHis system : six histidine residues are tagged tothe recombinant protein. The histidine tag and protein can be purified usinga nickel-chelating resin.

• Example 2 : pMal system : tags = maltose binding protein to therecombinant protein. The maltose binding protein tag will bind strongly to anamylose column.

Brand Name Ligand Application ExamplesHiTrap Protein A Protein A IgG, IgG subclasses, fragments of

IgG, IgA, antigens, immune complexes

HiTrap Progein G Recombinant Protein G IgG, IgG subclasseslacking the albumin binding region

HiTrap Heparin Heparin Growth factors, coagulation proteins,plasma proteins, lipases, lipoproteins,enzymes that act on nucleic acids,steroid receptors, protein synthesisfactors

HiTrap Chelating Iminodiacetic acid Proteins and peptides with exposedhistidine groups

HiTrap Blue Cibacron Blue F3GA Enzymes requiring adenyl-containingcofactors, albumin, coagulationfactors, interferon

Brand Name Ligand ApplicationsAffi-Gel Protein A Protein A Purification of IgG from acites, serum andAffi-Prep Protein A culture fluid.

Affi-Gel Blue Cibacron Blue F3GA Binds many nucleotide-requiring enzymes,albumin and other proteins.

DEAE Affi-Gel Blue Cibacron Blue F3GA Binds albumin and serum proteins and DEAE groups. Used to purify protease-free IgG from acites,

serum and cutlure fluid

Affi-Gel heparin Heparin Purification of a wide variety of proteinsincluding growth factors, coagulation factors,DNA and RNA specific enzymes, lipase,lipoproteins and proteases

Affi-Prep polymyxin Polymixin Endotoxin removal

Affi-Gel 501 Organomercurial Adsorbtion of sulfhydrl proteins and low mwsulfhydrls via thiol groups. Bound proteins areeluted with dilute mercaptoethanol or dithioreitol.

Affi-Gel 601 Boronate Adsorbtion of cis-hyroxyl containing moleculesincluding sugars, nucleotides and glycocpeptides.



PROTEIN STAINING

PROTEIN STAINING• Amidoblack• Commassie• Ponceau-red• Silver• Gold• Gelcode

Coomassie Blue Dyes - commonly used- does not interfere with subsequent protein identification- inexpensive- sensitivity well below silver and fluorescent dyes

Silver stain- sensitivity 10-50 times greater than CB- ability to detect 1 ng of protein- silver diammine/silver nitrate- relatively expensive (reagents/waste disposal)- high background

Fluorescent Stains and Dyes- accurately determine changes in protein expression- greater sensitivity than silver stain- DIGE- cost

Protein Imaging

Silver-stained two-dimensional polyacrylamide-gel

PROTEIN STAININGAmidoblack

spot 5 ul protein sample on nitrocellulose (NC)- air-dry for 5 min- immerse in stain solution for 3 min

- wash 2x 3 min in water- wash 2x 3 min in wash solution- wash 5 min with water- air-dry 5 min

- elute stain with 1 ml elution solution (while shaking the sample)

- measure absorption at 630 nm of the eluant

PROTEIN STAININGAmidoblack

stain = 0.1 % amidoblack, 45% methanol, 10% acetic acid

wash = 90% methanol, 2% acetic acid

elution = 50% ethanol, 50% 50 mM NaOH/0.1 mM EDTA

- use BSA solutions (0 to 5 mg/ml) for calibration

- this protocol also works for proteins in SDS-PAGE buffer

PROTEIN STAININGCoomassie Blue

• 1. Immerse gel in 50% ethanol/10% acetic acid for at least 1 hr.

• 2. Soak in 5% ethanol/5% acetic acid overnight or for aminimum of 2 hours.

• 3. Wash in diH2O for 1 hr.• 4. Add Gel-Code Blue Stain reagent for at least 3 hrs.

(Pierce, #24592)• 5. Wash in diH2O twice, 15 min each.• 6. Rinse in diH2O for 1 hr.• 7. Gels can be stored at 4°C.

PROTEIN STAININGCoomassie-Blue

&Amidoblack

PROTEIN STAININGPonceau Red

• For Membrane after Blotting. To check blotting• Reversible: gets washed out with destaining

solution

• Ponceau S stock solution: 200 mg Ponceau S per100 ml 3 % Perchloric acid

• ready-to-use solution : dilute stock sol. 1:5 with10% acetic acid

• Background destaining :10 %acetic acid

PROTEIN STAININGSilver

PROTEINSTAINING

OTHER STAINS BioRad-Zinc

Gold-Stain Silver-Stain Coomassie-BlueGold-Blot Sypro-Orange Sypro-Red



IMMUNOPRECIPITATION

IMMUNOPRECIPITATIONAPPLICATIONS-I

Immunoprecipitation can be used for many purposes :• 1) Determination of the molecular weight and

isoelectric point of immunoprecipitated proteins byone-dimensional or two-dimensional SDS-PAGE.

• 2) Verification that an antigen of interest issynthesized by a specific tissue (i.e., that radiolabeledprotein can be identified in tissues or cells culturedwith radiolabeled precursors).

• 3) Determination of whether a protein containscarbohydrate residues by evaluating whetherimmunoprecipitated antigen from cells cultured withradioactive monosaccharides is radiolabeled.

IMMUNOPRECIPITATIONAPPLICATIONS - II

• 4) Characterization of the type of carbohydratepresent on glycoproteins - evaluate incorporation ofdifferent radiolabeled monosaccharides intoimmunoprecipitated protein during cell culture and testwhether inhibitors of glycosylation alter the molecularweight of immunoprecipitated protein.

• 5) Determination of precursor-product relationships byperforming pulse-chase labeling followed byimmunoprecipitation.

• 6) Quantification of synthesis rates of proteins inculture by determining the quantity ofimmunoprecipitated, radiolabeled protein.

Immunoprecipitation

IP - anti HAprobe - α p-VEGFR

Mixedproteins

Primary(mouse α-HA)

Secondary(rabbit α-mouse)

IP - anti HAprobe - anti shc

Primary sheep α ShcSecondary donkey α sheep

= HA-VEGFR (Haemagglutinin)

= shcImmuno-co-precipitation

Addantibody

Agarose bead conjugatedto secondary antibody

Addbeadedαbody

Spin

resuspend pelletand load on gel

Warner et al Biochem. J. (2000) 347, 501–509

VEGF

VEGF

IP anti HA

IP anti HA

Kinase assays

1. Add radioactive phosphateand substrate

2. Subject to SDS PAGE

32P

32P

Unincorporated32P

32P labelledsubstrate

3. Develop against film

PI3 Kinase inhibitorsVehicle

Stimulant

Porcine aortic endothelial cells tranfected with VEGF-R2

Qi & Claessen-Welsh, Exp Cell Res 263 173-182 (2001)

IMMUNOPRECIPITATION

Explants cultured in the presence of[35S]met incubated w. Ab to uterine milkprotein (UTMP).

Ag-Ab absorbed w. Protein A-SepharoseSDS-PAGE + fluorography.

Ab = proteins immunoprecipitated withrabbit antiserum to UTMP

NRS = normal rabbit serum TC = total array of radiolabeled proteins

present in the unabsorbed sample

IMMUNOPRECIPITATIONSOME PROBLEMS I

• 1) crossreactivity : attention must be given toantibody cross-reactivity with other antigens (like allimmunochemical procedures)

• 2) Nonspecific binding : can be a problemespecially if proteins that are immunologically distinctfrom the antigen are trapped in the pellets formedduring immunoprecipitation.

IMMUNOPRECIPITATION• To reduce nonspecific binding, immuno-precipitation

buffers usually have– some detergent to reduce hydrophobic interactions,– a protein to block nonspecific binding sites,– and high salt to reduce ionic interactions.

• In many protocols, a preclearing step is performedto remove molecules that nonspecifically bind toinsoluble Protein A or Protein G.

• Despite these precautions, nonspecific binding canoccur.

IMMUNOPRECIPITATIONIt is crucial, therefore, to always perform a control

reaction where antibody is replaced by a non-relevantimmunoglobulin (i.e, normal serum for polyclonalantibodies, control mouse ascites fluid for ascites, andisotype controls for purified mouse monoclonalantibodies).

IMMUNOPRECIPITATIONSOME PROBLEMS - II

• 3) Proteolytic digestion : can occur when cells are lysedand contents of lysosomes are mixed with other compartmentsof the cell.Accordingly, most immunoprecipitation buffers containproteinase inhibitors.

• 4) Care should be taken in using immunoprecipitation as aquantitative tool to determine rates of synthesis of proteinsbecause the rate of incorporation of radiolabel into protein willdepend upon rate of synthesis of a protein as well as the rate ofdilution of radiolabeled precursor by the intercellular pool ofprecursor.

IMMUNOPRECIPITATIONSOME PROBLEMS - III

• 5) Protein Trapping : molecules too small or too large to beresolved by SDS-PAGE are sometimes trapped in the pelletformed by immobilized Protein A or Protein G.

• These molecules, while not interfering with analysis by SDS-PAGE, canmake direct quantification of radiolabeled antigen by scintillationspectrometry problematic.

• Thus, immunoprecipitated protein should be quantified bydensitometric analysis of autoradiographs or fluorographs.Scintillation spectrometry sometimes reveals little difference in thequantitative yield of radioactivity between an immunoprecipitationreaction and a control reaction (i.e., where antibody has beensubstituted with normal rabbit serum). Nonetheless, subsequentanalysis by SDS-PAGE reveals precipitation of radiolabeled protein inthe antibody reaction only.

IMMUNOPRECIPITATION

SOME PROBLEMS - IV

• 6) Sensitivity : can be a problem,especially when the antigen is a minor component of theprotein pool.New screen technology for low energy radioisotopes (TranscreenLE enhancing screens by Kodak) increases sensitivity greatly.

Use as much protein in the immunoprecipitation reaction aspossible.

III. FASPS (4)Serine to glycine mutation in hPER2 of FASP person

Fig 13. Mapping of the CKIε binding domain of PER2 by immunoprecipitation(Toh et al, 2001).

<Experimental scheme>

PER2 mycCKIε

rabbitreticulocyte

mper2 cDNA

ck1ε cDNA

anti-MYCantibody

PER2 mycCKIε

Immuno-precipitation

Separation in SDS PAGE gels

Fig 14. In vitro CKIe phosphorylation of wildtype and mutant hPER2(Toh et al, 2001).

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METABOLIC LABELLING

• Aims:

An important measurement ofAn important measurement ofmetabolism in metabolic engineering ismetabolism in metabolic engineering isin vivoin vivo metabolic flux: metabolic flux: the rate of flow ofthe rate of flow ofbiochemical material down a pathway.biochemical material down a pathway.

A B C D

Rates expressed in units of quantity for unit time per unit tissue mass e.g. Rates expressed in units of quantity for unit time per unit tissue mass e.g. nmolnmol.min.min-1-1.g.g-1-1 FwFw

METABOLIC LABELLING• Aims

:

Metabolic Flux Analysis (MFA)Metabolic Flux Analysis (MFA)

Stoichiometric Stoichiometric flux balance analysisflux balance analysisIsotopic flux balance analysis :Isotopic flux balance analysis :

radiolabelingradiolabeling

stable isotope labelingstable isotope labelingsteady-state

non-steady-statenon-steady-state

steady-state

All types of MFA involve algebraic representationsAll types of MFA involve algebraic representationsof metabolism with numerous interacting variables. Effective handling ofof metabolism with numerous interacting variables. Effective handling ofthese interacting variables requires computer simulation modelsthese interacting variables requires computer simulation models..

dynamicdynamickinetickineticmodelsmodels

steady-state

Principles of flux determination fromPrinciples of flux determination fromradiolabeling radiolabeling kineticskinetics

M*

M

(external radiolabeled M)

J J

The flux, J, can be determined by introducing apulse of radiolabeled M (M*) and measuring theradioactivity in samples of purified M.

Stephanopoulos, G.N., Aristidou, A., Nielsen, J. 1998. “Metabolic EngineeringPrinciples and Methodologies”. Academic Press, San Diego.

dM* M*dt M= - J

The pool of intracellular radiolabeledM will change according to thefollowing equation:

Radiolabeling Experiments1. Labeled metabolite of known specific activity supplied2. Extraction of leaf tissue3. Phase separation4. Ion exchange chromatography5. Thin layer chromatography or electrophoresis6. Quantify metabolites by scintillation counting

14C, 33P-labeled precursor

Practical concernsIs the supplied labeled compound taken up?Is it altered before uptake ? (e.g., phosphorylated compounds)Can you separate all relevant metabolites?Are the metabolites efficiently recoverable?

Leaf disk labeling

Advantages of RadiolabelingExperiments

• Very high sensitivity- can quantify turnover in small pools- only small tissue quantities are required; replication,

many time points possible fore kinetic studies

• Straightforward quantification (scintillation counting)- must account for quenching (pigments, solvents)- metabolites must be separable so that label in different

compounds not counted together.- metabolites not labeled via intersecting pathways- need to consider the potential for labeling of multiple atoms

• High time resolution- critical for rapid kinetics

METABOLIC LABELLINGHow long should cells be labeled?• The ideal length of time to label cells depends on the protein of

interest and the label that you are using.• If you want to label an unstable protein with 35S-methionine, a

short labeling interval--no more than 2 hr—is best.• If you are studying a stable protein, a longer label may be

preferable.• The issue is the half-life of the protein of interest relative to the

half-life of the background bands. The half-life of total cellularprotein is 45 to 50 hr.

METABOLIC LABELLINGLabeling with 32Pi is different.• the phosphate in proteins undergoes continual turn-over in

most cases. Therefore, both old and newly-synthesizedproteins become labeled soon after 32Pi is added to the cells.

• A short labeling period with 32Pi is advantageous : labeling of RNA and DNA is less obvious than in cells labeled over-night.

• For studying tyrosine protein kinases: phosphotyrosines tend toturn-over faster than the bulk of either phosphoserine or phosphothreonineand thus are preferentially labeled during brief labeling.

METABOLIC LABELLING

• over-night labeling period is optimal if looking atthe steady-state abundance of phosphate in proteins, lipids, orRNA,

• Under these conditions, the specific activity of the ATP pool inthe cell and of the phosphates in macromolecules is beginningto approach that of the medium.

• Additionally, the amount of label present in proteins, lipids andRNA should now reflect the amount of phosphate present,rather than the rate of turn-over of the phosphate.

METABOLIC LABELLING

• An issue to consider : radiation damage !!!• Can induce the stabilization of p53 and cause cell cycle arrest.• Cells labeled for a prolonged period with 32P may therefore not

be growing when you harvest them.• the specific activity of the ATP pool comes to equilibrium with

the phosphate in the medium by 6 hours. Therefore there is noreason to label for longer with 32Pi.

METABOLIC LABELLING• Overnight labeling is best done in medium containing a

reduced concentration of phosphate or methionine. 10% isoften reasonable, depending on the cell line.

• easily accomplished by using methionine-free or phosphate-free medium and 10% undialyzed serum (which can be assumed tocontain the same concentration of methionine or phosphate as normal medium).

METABOLIC LABELLING• For short term labeling (30 seconds to 5 hrs)

Use :• (1) medium completely lacking either phosphate or methionine,• (2) serum which has been dialyzed against saline, and• (3) a fairly low volume of medium;• 0.75 ml for a 35 mm dish, 2 to 2.5 ml for a 50 mm dish, and 2.5 to 5 ml

for a 100 mm dish.

• It is a good idea to rinse the cells you are going to label withlabeling medium—which lacks label—prior to adding the actuallabeling medium.

• Starvation doesn't help much.

Documents

MODERN METHODS in BIOCHEMISTRY - unifr.ch · modern methods in biochemistry •protein modification •protein crosslinking •protein staining •antibody modification •immunoprecipitation