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ANALYSIS OF PROTEINS BY IMMUNOPRECIPITATION

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ANALYSISOFPROTEINSBYIMMUNOPRECIPITATION

P.J.Hansen

Dept.ofAnimalSciences,UniversityofFlorida

INTRODUCTION

Immunoprecipitationisaprocedurebywhichpeptidesorproteinsthatreactspecificallywithanantibodyareremovedfromsolutionandexaminedforquantityorphysicalcharacteristics(molecularweight,isoelectricpoint,etc.).Asusuallypracticed,thenameoftheprocedureisamisnomersinceremovaloftheantigenfromsolutiondoesnotdependupontheformationofaninsolubleantibody-antigencomplex.Rather,antibody-antigencomplexesareremovedfromsolutionbyadditionofaninsolubleformofanantibodybindingproteinsuchasProteinA,ProteinGorsecondantibody(Figure1).Thus,unlikeothertechniquesbasedonimmunoprecipitation,itisnotnecessarytodeterminetheoptimalantibodydilutionthatfavorsspontaneously-occurringimmunoprecipitates.

Figure1.Schematicrepresentationoftheprincipleofimmunoprecipitation.AnantibodyaddedtoamixtureofrADIolabeled(*)andunlabeledproteinsbindsspecificallytoitsantigen(A)(lefttube).Antibody-antigencomplexisabsorbedfromsolutionthroughtheadditionofanimmobilizedantibodybindingproteinsuchasProteinA-Sepharosebeads(middlepanel).Uponcentrifugation,theantibody-antigencomplexisbroughtdowninthepellet(rightpanel).SubsequentliberationoftheantigencanbeachievedbyboilingthesampleinthepresenceofSDS.

Typically,theantigenismaderadioactivebeforetheimmunoprecipitationprocedure,eitherbyculturingcellswithradioactiveprecursororbylabelingthemoleculeaftersynthesishasbeencompleted(e.g.,byradioiodinationtoiodinatetyrosineresiduesorbysodium[³H]borohydridereductiontolabelcarbohydrate).Havingaradioactiveantigenisnotrequiredbutinterpretationofdataaresimplifiedsincetheantigen,andnottheantibody,isradiolabeled.Analysisoftheimmunoprecipitateisusuallybyelectrophoresisalthoughothertechniquescanbeused.

Thechoiceofimmobilizedantibodybindingproteindependsuponthespeciesthattheantibodywasraisedin.ProteinAbindswelltorabbit,cat,human,pigandguineapigIgGaswellasmouseIgG_2aandIgG_2b.ProteinGbindsstronglytoIgGfromcow,goat,sheep,cow,horse,rabbitandguineapigandtomouseIgG₁andIgG₃.ProteinGcanalsobindbovineserumalbumin(BSA).Thus,BSAshouldbeaddedtobuffersusedwithProteinG.Alternatively,recombinantProteinGwithoutBSAbindingsitescanbeused(ProteinGPlusfromOncogeneScience).

APPLICATIONS

Immunoprecipitationcanbeusedformanypurposes.Amongtheseare:

1)Determinationofthemolecularweightandisoelectricpointofimmunoprecipitatedproteinsbyone-dimensionalortwo-dimensionalSDS-PAGE.

2)Verificationthatanantigenofinterestissynthesizedbyaspecifictissue(i.e.,thatradiolabeledproteincanbeidentifiedintissuesorcellsculturedwithradiolabeledprecursors).

3)Determinationofwhetheraproteincontainscarbohydrateresiduesbyevaluatingwhetherimmunoprecipitatedantigenfromcellsculturedwithradioactivemonosaccharidesisradiolabeled.

4)Characterizationofthetypeofcarbohydratepresentonglycoproteins-evaluateincorporationofdifferentradiolabeledmonosaccharidesintoimmunoprecipitatedproteinduringcellcultureandtestwhetherinhibitorsofglycosylationalterthemolecularweightofimmunoprecipitatedprotein.

5)Determinationofprecursor-productrelationshipsbyperformingpulse-chaselabelingfollowedbyimmunoprecipitation.

6)Quantificationofsynthesisratesofproteinsinculturebydeterminingthequantityofimmunoprecipitated,radiolabeledprotein.

PROCEDURES

Thefollowingaretwomethodsthathavebeenusedinourlaboratory.Botharesimilarinmanyrespects.Otherprotocolswillalsogivegoodresults.

METHODI

A.Reagents

IMPBuffer¹50mMTris-acetate,pH7.5²1mMphenylmethylsulfonylfluoride(PMSF)³1mMEDTA0.3MNaCl1mg/mlBSA2%IGEPALCA-630(v/v)⁴0.02%NaN₃Storeat4Cindefinitely

WashingBuffer50mMTris-acetate,pH7.50.3MNaCl0.5%IGEPALCA-6300.1%sodiumdodecylsulfate(SDS)⁵0.02%NaN₃Storeat25Cindefinitely

ProteinA-SepharoseCL-4-BSUSPension⁶ProteinA-Sepharosebeads(PharmaciaorSigma)areswelledinPBSorIMPbufferonatubeturneratroomtemperature.BeadsarethenwashedtwiceinIMPbufferanddilutedto10%(v/v)inIMPbuffer.Dilutioncanbeperformedbyestimatingtheapproximatevolumeofswollenbeadsinagraduatedconicalcentrifugetubeandadding9volumesofbuffer.Beadscanbestoredat4Cindefinitely.

Notes:1)StockscanbemadeofTris-acetate(1M),EDTA(0.2M),PMSF(0.1Minethanol)andNaN₃(20%,v/v)tosimplifymakingupbuffers.

2)NotethatTrisbuffersareverytemperature-sensitiveandshouldbepreparedusingwateratthetemperaturetheywillbeusedat.OtherbufferssuchasphosphatecanbeusedinsteadofTris.

3)PMSFisverylABIleinwater.Amoreexpensivebutmorestablealternativeis4-(2-aminoethyl)-benzenesulfonylfluoridehydrochloride(AEBSF;Pefabloc®)fromBoehringer-Mannheim(0.1-0.5mg/ml).InadditiontoPMSFandEDTA,otherproteinaseinhibitorscanalsobeincluded(forexample,10µg/mlleupeptinand0.7µg/mlpepstatin).TheBoehringerMannheimcatalogcontainsaverydetailedsummaryofproteinaseinhibitorsavailableforuse.

4)TheprotocolwasoriginallyusedwithNonidetP-40,whichisnolongeravailable.IGEPALCA-630fromSigmaischemicallyindistinguishablefromNonidetP-40.

5)SDStendstoprecipitateat4Calthoughlowconcentrationsusedheremaynotbeaproblem;canreplacewithanequalamountofsarkosylifitisdesiredtostoreintherefrigerator.

6)SecondantibodycoupledtoSepharoseorProteinG-Sepharosecanbeusedinstead.ItisalsonotcrucialthatSepharosebeusedasamatrix.OtherpolymerizedagarosesorevenfixedstrainsofStaphylococcuscellsexpressinghighamountsofsurfaceProteinAcanbeused.

B.ProcedureNote:Theprocedurehereassumesthataconcentrationstepisrequiredtoobtainenoughradioactivityfortheimmunoprecipitationanalysis.Otherproceduresforconcentrationotherareavailablebesidesthedialysis/lyophilizationproceduredescribedhere(forexample,useofCentricondevicesfromAmicon).Forsomeapplications,samplescanbeanalyzedwithoutconcentrationandafterdilutionwithimmunoprecipitationbuffer.

1)Prepareradiolabeledproteinmixturebyculturingcellswithradiolabeledprecursor,radioiodinationorothermethod.

2)Forsolubleantigens(i.e.,conditionedmedium),dialyzesampleagainstwater.Forintracellularantigen,harvestcells,andlysecellswithappropriatedetergent.Somethathavebeenusedinclude1%(v/v)TritonX-100,IGEPALCA-630,Renex30,orCHAPS.Ifdialysisistobeusedtoconcentrateprotein,useadialyzabledetergent(example,deoxycholateorCHAPS).Thebufferusedforlysisshouldcontainproteinaseinhibitors(forexample,PMSF,EDTA,aprotinin,andleupeptin).

Figure2.Representativeresultfromimmunoprecipitation.Conditionedculturedmediumfromendometrialexplantsculturedinthepresenceof[³⁵S]methioninewasincubatedwithanantibodytoanendometrial-specificproteincalleduterinemilkprotein(UTMP).Antibody-antigencomplexeswereabsorbedusingProteinA-SepharoseandanalyzedbySDS-PAGEandfluorography.LanesrepresentproteinsimmunoprecipitatedwithrabbitantiserumtoUTMP(Ab),normalrabbitseruminplaceofantiserum(NRS)andthetotalarrayofradiolabeledproteinspresentintheunabsorbedsample(TC).FromLeslieandHansen(1991).

3)Foreachsampletested,lyophilize2aliquantsof200,000dpmofradiolabeledproteinand1aliquantof100,000dpmin1.5mlmicrocentrifugetubes.Thesamplesof200,000dpmwillbeusedforimmunoprecipitationwithantibodyandcontrolimmunoglobulinandthe100,000dpmtube(optional)willbeusedtodeterminetheentirearrayofradiolabeledproteinspresentinthesample.

Note:Todetermineradiolabeledprotein,onemustremoveunincorporatedradiolabelfirst.Thiscanbedonebydialysisorgelfiltration.Alternatively,incorporationofradiolabelcanbedeterminedbytrichloroaceticacidprecipitation.Lowerdpmthanstatedherecanbeusedifneededbutsensitivitywilldecrease.Wehaveusedasmanyas6milliondpmtomaximizesensitivity.

4)Putthe100,000dpmsamplesasideuntilstep#12.

5)Afterlyophilization,solubilize200,000dpmsamplesin350µlIMPbuffer.

6)Add50µlofantibodyorcontrol(normalrabbitserum,mouseisotypecontrol,,etc.)toeachtubeandincubateovernightfor6hatroomtemperature.

7)Add100µlofProtein-A-Sepharosesolution(shaketosuspendslurrybeforepipetting)andincubateonatubeturnerfor6hatroomtemperature.

8)Centrifugeof1minonmicrocentrifugeandsavepellet.

9)Add500µlwashingbuffer,vortex,spin1minonmicrocentrifugeanddiscardsupernatant.

10)Add1mlwashingbuffer,vortex,spin1min,discardsupernatant;repeat3x.

11)Add1ml10mMTris-acetate,pH7.5,vortex,spin1min,discardsupernatant.

12)Solubilizeallsamples(including100,000dpmsamples)in30-50µlofthegelsolubilizationbufferusedforSDS-PAGE,boilfor3minandcentrifuge.

13)SavesupernatantandanalyzebySDS-PAGEandfluorographyorautoradiography(¹²⁵I)orfluorography(³H,¹⁴C,and³⁵S).

MethodII

A.Reagents

IMPBuffer50mMTris-acetate,pH7.5150mMNaCl1%(v/v)TritonX-1001%(w/v)deoxycholate0.1%(w/v)SDS4mMAEBSF1µg/mlaprotinin¹10µg/mlleupeptin¹0.02%(w/v)NaN₃

Notes:1)Preparefroma1mg/mlstockinwater.Storefrozeninaliquots.

ProteinGPlus-AgaroseFromOncogeneScience(Cat#IP04).Thisproduct,whichisreadytouse,is30%(v/v)agarosebyvolumeandcontains20mgIgGbindingcapacity/mlpackedbeads.

Procedure1)PreparesamplesasforProcedureI.Foreachsampletested,prepare2aliquantsof200,000dpmeachand1aliquantof100,000dpmin1.5mlmicrocentrifugetubes.

2)Putthe100,000dpmsamplesasideuntilstep#11.

3)Afterlyophilization,solubilize200,000dpmsamplesin300µlIMPbuffer.

4)Topreclearsamples,add50µlofcontrol(normalrabbitserum,mouseisotypecontrol,etc.)toeachtubeand25µlofProteinGslurry(shaketosuspendslurrybeforepipetting)andincubatefor1hat4C.

5)Centrifugeof1minonmicrocentrifugeandsavesupernatant.

6)Addantibodyorcontrol(50µlmaximumvolume,dilutedinIMPbuffer)andincubate1hatroomtemperature.

7)AddProteinGagarose(25µl)overnightat4Conatuberotator.

8)Centrifuge1minonmicrocentrifuge,discardsupernatant.

9)Add1mlIMPbuffer,vortex,spin1min,discardsupernatant;repeat3x.

10)Add1ml10mMTris-acetate,pH7.5,vortex,spin1min,discardsupernatant.

11)Solubilizeallsamples(including100,000dpmsamples)in30-50µlofthegelsolubilizationbufferusedforSDS-PAGE,boilfor3minandcentrifuge.

12)SavesupernatantandanalyzebySDS-PAGEandfluorographyorautoradiography(¹²⁵I)orfluorography(³H,¹⁴C,and³⁵S).

SOMEPROBLEMS1)Likeallimmunochemicalprocedures,attentionmustbegiventoantibodycrossreactivitywithotherantigens.

2)Nonspecificbindingcanbeaproblemespeciallyifproteinsthatareimmunologicallydistinctfromtheantigenaretrappedinthepelletsformedduringimmunoprecipitation.Toreducenonspecificbinding,immuno-precipitationbuffersusuallyhavesomedetergenttoreducehydrophobicinteractions,aproteintoblocknonspecificbindingsites,andhighsalttoreduceionicinteractions.Inmanyprotocols,apreclearingstepisperformedtoremovemoleculesthatnonspecificallybindtotheinsolubleProteinAorProteinG.Despitetheseprecautions,nonspecificbindingcanoccur.Itiscrucial,therefore,toalwaysperformacontrolreactionwhereantibodyisreplacedbyanon-relevantimmunoglobulin(i.e,normalserumforpolyclonalantibodies,controlmouseascitesfluidforascites,andisotypecontrolsforpurifiedmousemonoclonalantibodies).

3)Proteolyticdigestioncanoccurwhencellsarelysedandcontentsoflysosomesaremixedwithothercompartmentsofthecell.Accordingly,mostimmunoprecipitationbufferscontainoneormoreproteinaseinhibitors.

4)Careshouldbetakeninusingimmunoprecipitationasaquantitativetooltodetermineratesofsynthesisofproteinsbecausetherateofincorporationofradiolabelintoproteinwilldependuponrateofsynthesisofaproteinaswellastherateofdilutionofradiolabeledprecursorbytheintercellularpoolofprecursor.

5)Ithasbeentheauthor"sexperiencethatsometimesscintillationspectrometryrevealslittledifferenceinthequantitativeyieldofradioactivitybetweenanimmunoprecipitationreactionandacontrolreaction(i.e.,whereantibodyhasbeensubstitutedwithnormalrabbitserum).Nonetheless,subsequentanalysisbySDS-PAGErevealsprecipitationofradiolabeledproteinintheantibodyreactiononly.Itislikely,therefore,thatmoleculestoosmallortoolargetoberesolvedbySDS-PAGEaresometimestrappedinthepelletformedbyimmobilizedProteinAorProteinG.Thesemolecules,whilenotinterferingwithanalysisbySDS-PAGE,canmakedirectquantificationofradiolabeledantigenbyscintillationspectrometryproblematic.Thus,immunoprecipitatedproteinshouldbequantifiedbydensitometricanalysisofautoradiographsorfluorographs.

6)Sensitivitycanbeaproblem,especiallywhentheantigenisaminorcomponentoftheproteinpool.Newadvancesinenhancingscreentechnologyforlowenergyradioisotopes(TranscreenLEenhancingscreensbyKodak)shouldincreasesensitivitygreatly.Intheauthor"slab,effortsaremadetouseasmuchproteinintheimmunoprecipitationreactionaspossIBLe.

BIBLIOGRAPHY

FurtherReadingCelis,J.E.,Lauridsen,J.B.,andBasse,B.(1994)DeterminationofantibodyspecificitybyWesternblottingandimmunoprecipitation.In:Celis,J.E.(ed.),CellBIOLOGy.ALaboratoryHandbook,AcademicPress,NewYork,Vol.2,pp.305-313.

Howlett,S.K.(1987)Qualitativeanalysisofproteinchangesinearlymousedevelopment.In:Monk,M.(ed.),MammalianDevelopment:APracticalApproach.IRLPress,Oxford,pp.163-181.

Mason,D.W.,andWilliams,A.F.(1986)Kineticsofantibodyreactionsandtheanalysisofcellsurfaceantigens.In:Weir,D.M.,Herzenberg,L.A.,Blackwell,C.,andHerzenberg,L.A.(ed.),HandbookofExperimentalImmunology,Blackwell,Oxford,vol.1,chapter38.

Sugimoto,K.(1994)DNAimmunoprecipitation:applicationtocharacterizationoftargetsequencesforahumancentromereDNA-bindingprotein(CENP-B).In:Celis,J.E.(ed.),CellBiology.ALaboratoryHandbook,AcademicPress,NewYork,pp.335-343.

ExamplesofApplicationsfromtheAuthor"sLaboratoryHansen,P.J.,Ing,N.H.,Moffatt,R.J.,Baumbach,G.A.,Saunders,P.T.K.,Bazer,F.W.,andRoberts,R.M.(1987)Biochemicalcharacterizationandbiosynthesisoftheuterinemilkproteinsofthepregnantsheeputerus.Biol.Reprod.36,405-418.

Helmer,S.D.,Hansen,P.J.,andThatcher,W.W.(1988)Differentialglycosylationofthecomponentsofthebovinetrophoblastprotein-1complex,novelglycoproteinssecretedbytheday17-18conceptus.Mol.Cell.Endocrinol.58,103-107.

Leslie,M.V.andHansen,P.J.(1991)Progesterone-regulatedsecretionoftheserpin-likeproteinsoftheovineandbovineuterus.Steroids56,589-597.

Plante,C.,Hansen,P.J.,Mirando,M.A.,Thatcher,W.W.andBazer,F.W.(1990)Developmentofantibodiesforstudyingconceptusinterferonsinthecow.J.Reprod.Immunol.18,205-223.

Edwards,J.L.,Ealy,A.D.,Monterroso,V.H.,andHansen,P.J.(1997)Ontogenyoftemperature-regulatedheatshockprotein70synthesisinpreimplantationbovineembryos.Mol.Reprod.Dev.,48,25-33.