NUNC
来自 : 蚂蚁淘
CO-IMMUNOPRECIPITATIONANALYSISOFPROTEIN-PROTEININTERACTIONS
Thisprotocolusestotalcellextractstoanalyzeputativeprotein-proteininteractionsineukaryoticcells.Onecanalsousenuclearextractsasasourceforthisprotocol.
- Transfect2-T150sofHEK293cellswiththeappropriateexpressionvectors(proteinAandproteinB)asperstandardprotocol.Alternatively,youcanuseanendogenouscelllinethatexpressedbothproteinsifyouhavespecificantibodiesforeachprotein.
- After48hours,harvestcellsandwashwithPBS-/-
- Transfercellsinto2microfugetubesandlyseeachtubeofcellswith1mlof:
20mMTrispH7.5
100mMNaCl
0.5%NP-40
0.5mMEDTA
0.5mMPMSF
0.5%proteaseinhibitorcocktail(Sigma)
pipetcellsupanddownextensivelyandthensitonicefor10minutes.
- Spindowncellextractsatmaxspeedfor10minutesat40C.
- Poolextractsfromeachtransfectionintoa15mltube.Forimmunoprecipitation,add10ml(polyclonalserum)or10mgs(monoclonalantibody)to1mlofextract.Incubatewithend-over-endmixingfor2hoursat40C.Save50mlofeachcellextractandadd50mlof2XSDS-PAGEsamplebufferandboilfor5min.
- Wash50mlofProteinA/GSepharose(SantaCruzBiotech)with1mloflysisbuffer---3timesandthenremovelastofexcessbuffercarefully.
- Afterthe2hourincubation(Step5),addthewashedProteinA/GSepharosetotheimmunoprecipiatesandmixend-over-endfor1hourat40C.
- Washimmunoprecipitates3Xwith1mloflysisbufferandremovelastofbuffercarefully.Add50mlof2XSDS-PAGEsamplebufferandboil5min.LoadonSDS-PAGEgelasfollows:
Lane1PAGEstandards
Lane2proteinAtransfectedcells
Lane3proteinBtransfectedcells
Lane4proteinA/Btransfectedcells
Alsorunagelwithjusttheextract(Step5)beforeimmunoprecipitationtoshowthateachcelllineexpressestherightprotein.
- WesternblotgettoImmobilonmembranes(Millipore)andblockinthefollowingbufferovernight:
Blotto
50mMTrispH7.5
100mMNaCl
0.1%Tween20
5%drymilk
- ProbethenextdaywiththeappropriateantibodycombinationanddevelopusingtheECLkit(seeWesternblotprotocol)
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