1.Washadherentcellstwiceinthedishorflaskwithice-coldPBSanddrainoffPBS.Washnon-adherentcellsinPBSandcentrifugeat800to1000rpminatable-topcentrifugefor5minutestopelletthecells.

2.Addice-coldmodifiedRIPAbuffertocells(1mlper10⁷cells/100mmdish/150cm²flask;0.5mlper5x106cells/60mmdish/75cm²flask).

3.Scrapeadherentcellsoffthedishorflaskwitheitherarubberpolicemanoraplasticcellscraperthathasbeencooledinice-colddistilledwater.TransferthecellsUSPensionintoacentrifugetube.Gentlymixthesuspensiononeitherarockeroranorbitalshakerat4for15minutestolysecells.

4.Centrifugethelysateat14,000xginaprecooledcentrifugefor15minutes.Immediatelytransferthesupernatantfractiontoafreshcentrifugetubeanddiscardthepellet.

5.ToprepareproteinAorGagarose/sepharose,washthebeadstwicewithPBSandrestoretoa50%slurrywithPBS.ItisrecommendedtocutthetipoffofthePipettetipwhenmanipulatingagarosebeadstoavoiddisruptionofthebeads.

6.Pre-clearthecelllysatebyadding100microlitersofeitherproteinAorGagarose/sepharosebeadslurry(50%)per1mlofcelllysateandincubatingat4for10minutesonarockerororbitalshaker.Pre-clearingthelysatewillreducenon-specificbindingofproteinstotheagaroseorsepharosewhenitisusedlateronintheassay.

7.RemovetheproteinAorGbeadsbycentrifugationat14,000xgat4for10minutes.Transferthesupernatanttoafreshcentrifugetube.

8.Determinetheproteinconcentrationofthecelllysate,e.g.byperformingaBradfordassay.Dilutethecelllysateatleast1:10beforedeterminingtheproteinconcentrationbecauseoftheinterferenceofthedetergentsinthelysisbufferwiththeCoomassie-basedreagent.

9.Dilutethecelllysatetoapproximately1mg/mltotalcellproteinwithPBStoreducetheconcentrationofthedetergentsinthebuffer.Amoreconcentratedcelllysate(i.e.,10mg/ml)maybenecessarytoimmunoprecipitateaproteinwhichisfoundinlowlevelsinacellmodel.

10.Addtherecommendedvolumeoftheimmunoprecipitatingantibody(seeCertificateofAnalysisfordetailedinformation)to500microliters(i.e.,500micrograms)ofcelllysate.Theoptimalamountofantibodythatwillquantitativelyimmunoprecipitatetheproteinofinterestshouldbeempiricallydeterminedforeachcellmodel.

11.Gentlymixthecelllysate/antibodymixtureforeither2hoursorovernightat4onarockeroranorbitalshaker.A2hourincubationtimeisrecommendedfortheimmunoprecipitationofactiveenzymesforkinaseorphosphataseassays.

12.Capturetheimmunocomplexbyadding100microlitersproteinAorGagarose/sepharosebeadslurry(50microliterspackedbeads)andgentlyrockingoneitherarockerororbitalshakerforeither1hourorovernightat4.Inmanyinstances,immunocomplexcapturecanbeenhancedbyadding2microgramsofabridgingantibody(e.g.,rabbit-anti-mouseIgG).ThisisespeciallyimportantwithantibodiesthatbindpoorlytoproteinAsuchasmouseIgG₁orantibodiesgeneratedinchicken.

13.Collecttheagarose/sepharosebeadsbypulsecentrifugation(5secondsinthemicrocentrifugeat14,000rpm).Discardthesupernatantfractionandwashthebeads3timeswith800microlitersice-coldmodifiedRIPAbuffer.Occasionally,washingwithmodifiedRIPAbufferwillstriptheimmunocomplexfromtheagarose/sepharosebeads.Inthesecases,washingwiththemilderPBSisrecommended.

14.Resuspendtheagarose/sepharosebeadsin60microliters2xsamplebufferandmixgently.Thiswillallowforsufficientvolumetorunthreelanes.

15.Theagarose/sepharosebeadsareboiledfor5minutestodissociatetheimmunocomplexesfromthebeads.ThebeadsarecollectedbycentrifugationandSDS-PAGEisperformedwiththesupernatantfraction.Alternatively,thesupernatantfractioncanbetransferredtoafreshmicrocentrifugetubeandstoredfrozenat-20forlateruse.Frozensupernatantfractionsshouldbereboiledfor5minutesdirectlypriortoloADIngonagel.

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