Antibody Precipitation with Protein A or G
来自 : 蚂蚁淘
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| Author:JamesTurmo |
| Source:ContributedbyJamesTurmo |
| Abstract:UsingImmobilizedProteinAorProteinGforImmunoprecipitation. |
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Purpose UsingImmobilizedProteinAorProteinGforImmunoprecipitation
Materials- ImmunoPure®ImmobilizedProteinA(Pierceproduct#20333)orImmunoPure®ImmobilizedProteinG(Pierceproduct#20398).
- Primaryantibody
- Immunoprecipitationbuffer:20mMsodiumphosphate,pH7.5,500mMNaCl,0.1%SDS,1%NP40,0.5%sodiumdeoxycholateand0.02%sodiumazide.>NOTE:50mMsodiumacetatebuffer,pH5.0,.500mMNaCl,0.1%SDS,1%NP-40and0.02%sodiumazidemaybeusedastheimmunoprecipitationbuffertoimprovetheefficiencyofbindingwithproteinG.
- ElutionBuffer:Pierce’sImmunoPure®IgGElutionBuffer(ProductNo.21004)or0.1Mglycine-HClbuffer,pH2.5.
- SDS-PAGEsamplebuffer(pH6.8)containing2%SDS,62.5mMTrisBase,10%glycerol,2-5%2-mercaptoethanol,orusePierceProductNo.39000.
Procedure NOTE:Performthefollowingexperimentinamicrocentrifugetube.
- Solubilizeantigenin50µlofimmunoprecipitationbufferandaddamolarexcessofprimaryantibodytotheproteinsolutioncontainingtheantigenofinterest.Adjustthevolumeofthesampleto0.2mlwithimmunoprecipitationbuffer.Typically,2-5µgofpurifiedantibodywillbesufficientforthisvolumeofprecipitationmix.
- Incubatethesampleovernightat4°C.
- AddappropriateamountofimmobilizedproteinAorGtotheantigen-antibodycomplex(~50µlofgelper5µgofantibody).
- Incubatethesamplewithgentlemixingfor2hoursatroomtemperature.WashtheimmobilizedproteinAorG-boundcomplexeswith0.5mloftheimmunoprecipitationbuffer,followedbycentrifugationfor2-3minutesinamicrocentrifuge.Discardthesupernatant.Repeatthiswashprocedureatleast6times.
- Elutetheboundantigen-antibodycomplexfromtheimmobilizedproteinAorGbyincubationfor5minuteswith50µloftheelutionbuffer.Centrifugethesample,collectthesupernatant,andincubatethegelwithanother50µloftheelutionbuffer(5minuteincubation).Then,centrifugethesampleandcollectthesupernatant.Combinethe2x50µlsupernatantsamples.
- ImmediatelyadjusttheproteinsampletoaphysiologicalpHbyadditionofasuitable,moreconcentratedbuffersuchas1.0MTris,pH7.5(10µlofthisbufferto100µlofthesupernatantshouldbesufficient).
- Desalttheelutedfraction.
- Thesampleisreadyforcharacterization.
Note IftheantigenistobecharacterizedbySDS-PAGE,Steps6-9maybeeliminated.Inthissituation,followtheaboveproceduretoStep5.UponcompletingStep5,washthegeloncewith0.5mldistilledwater.CentrifugetheimmobilizedproteinAorGfor2-3minutesanddiscardthesupernatant.Thenincubatethecomplex-boundimmobilizedproteinAorGfor5minutesat95°Cwith25µloftheSDS-PAGEsamplebuffer.Centrifugethesampleandcollectthesupernatant.Repeat,poolingsupernatantforatotalof50µl.Thesampleisnowready
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