ForProteinConcentrationDeterminationofCellCulture
- Decantmediumfrom10cmdishofadherentcellsandrinseplaterapidlywithphosphate-bufferedsaline(PBS).
- AspirateexcessPBS.
- Add1mlboilinglysisbuffer(1%SDS,1.0mMsodiumortho-vanadate,10mMTrispH7.4).
- Scrapecellsfromdish,transfertoamicrocentrifugetube,andboilforanadditional5minutes.Toreduceviscosity,thesamplemaybesonicatedbrieflyorpassedseveraltimesthrougha26-gaugeneedle.
- Centrifugethesamplefor5minutestopelletinsolublematerial,thendiscardpellet.
- Diluteanaliquotofthecelllysatesampleatleast10-foldfortheBCA(Pierce)proteinconcentrationassay(SDSconcentrationmustbebelow0.1%toavoidinterferencewiththecolorimetricreADIng).
ForProteinGelElectrophoresisofCellCulture(withoutdeterminingproteinconcentration)
- Decantmediumfrom10cmdishofadherentcellsandrinseplaterapidlywithphosphate-bufferedsaline(PBS).
- AspirateexcessPBS.
- Add1mlboiling2Xconcentratedelectrophoresissamplebuffer(125mMTrispH6.8,4%SDS,10%glycerol,0.006%bromophenolblue,1.8%beta-mercaptoethanol).
- Scrapecellsfromdish,transfertoamicrocentrifugetube,andboilforanadditional5minutes.Toreduceviscosity,thesamplemaybesonicatedbrieflyorpassedseveraltimesthrougha26-gaugeneedle.Centrifugethesamplefor10minutestopelletinsolublematerial.Discardpellet.
- Thecelllysatesample(supernatant)isnowreadyforloadingontoyourgel.
ForProteinConcentrationDeterminationofWholeTissue
- Rapidlyhomogenizeevery0.25gtissuein3.5mlofboilinglysisbuffer(1%SDS,1.0mMsodiumortho-vanadate,10mMTrispH7.4).
- Microwavefor10–15seconds.
- Centrifugethehomogenate(16,000xg,15C)for5minutestopelletinsolublematerial,thendiscardpellet.
- Diluteanaliquotofthetissuelysatesampleatleast10-foldfortheBCA(Pierce)proteinconcentrationassay.
Guidelinesforchoosingthepercentgeltobeusedforcertainmolecularweightproteins(basedon37:1acrylamide:bisacrylamideratio)
4-5%gels:>250kDa
7.5%gels:250-120kDa
10%gels:120-40kDa
13%gels:40-15kDa
15%gels:<20kDa
GelElectrophoresis
- Ifnotalreadyinelectrophoresissamplebuffer,addanequalvolumeof2Xsamplebuffer(125mMTrispH6.8,4%SDS,10%glycerol,0.006%bromophenolblue,1.8%b-mercaptoethanol)toallsamplesandboilfor3–5minutes.
- Apply5-20µgtotalproteinofcellortissuelysatetoeachwellofa0.75–1.0mmthickgel.Forthickergels(1.5mmthick),applyupto25-40µgineachwell.
- Electrophoreseuntilthebromophenolblueinthesamplesreachesthebottomofthegel.Turnoffpowersupply.Keepgelsinrunningbufferuntilreadytotransfer.
WetTransfer
Note:Sinceextranegativechargesareneededtoreach1Ampinawettransfersystem,adjustthepHofthetransferbuffertoapproximatelypH8.0usingNaOH.
- Fortransferofproteinssmallerthan20kDa,transferproteinsfromgeltoPVDF(polyvinylidenedifluoride)membraneat1Ampconstantcurrentfor45minsorequivalent(250mAmpfor3hoursor500mAmpfor90minutes)intransferbuffer(25mMTris,190mMglycine,20%MeOH).
- Fortransferofproteinssmallerthan120kDa,transferproteinsfromgeltoPVDFmembraneat1Ampconstantcurrentfor1hourorequivalent(250mAmpfor4hoursor500mAmpfor2hours)intransferbuffer(25mMTris,190mMglycine,20%MeOH).
- Forproteinslargerthan120kDa,transfertoPVDFmembraneat1Ampconstantcurrentfor90minutesorequivalent(250mAmpfor6hoursor500mAmpfor3hours)intransferbuffer+SDS(25mMTris,190mMglycine,20%MeOH,0.05%SDS).
- ForProteinslargerthan250kDa,transfertoPVDFmembraneat1Ampconstantcurrentfor1hourand45minutesorequivalent(500mAmpfor3.5hours)intransferbuffer+SDS(25mMTris,190mMglycine,20%MeOH,0.05%SDS).
Semi-DryTransfer
Fortransferofproteinsfrom10%or13%gelstoPVDFmembranessemi-drytransfercanalsobeused.TransferproteinstoPVDFmembraneat1.2mAmp/cm2for1hourand45minutesintransferbuffer(25mMTris,190mMglycine,20%MeOH).
Optional
Ifblotsarenottobeusedforcolorimetricdetection,visualizethetransferredproteinsbystainingthemembranefor15minuteswithIndiaink(HigginsblackIndiaink,EberhardFaber)diluted1:1000inwashbuffer(10mMTrispH7.5,100mMNaCl,0.1%Tween20).Rinseexcessstainwithwashbufferbeforeblocking.
FORALLANTIBODIESEXCEPTPHOSPHOTYROSINE
- 1.Removetheblotfromthetransferapparatusorstainingtrayandimmediatelyplaceintoblockingbuffer(5%non-fatdrymilk,10mMTrispH7.5,100mMNaCl,0.1%Tween20).
- 2.Incubatetheblotfor30minutesat37°C,1houratroomtemperature,orovernightat4°C.
FORPHOSPHOTYROSINEANTIBODIES
- Removetheblotfromthetransferapparatusorstainingtrayandimmediatelyplaceintoblockingbuffer(1%BSA,10mMTrispH7.5,100mMNaCl,0.1%Tween20).
- Incubatetheblotfor30minutesat37°C,1houratroomtemperature,orovernightat4°C.
NOTE:THEINCLUSIONOFSODIUMAZIDEISTOBEAVOIDEDINALLSTEPSUSINGHRPO(HORSERADISHPEROXIDASE)CONJUGATES.
- DiluteHRPOconjugatedantibodyinthecorrespondingblockingbuffer
- Decantblockingbufferfromtheblot,addthedilutedantibodysolutionandincubatewithagitationfor30minutesat37°C,onehouratroomtemperatureorovernightat4°C.
- Decantthehorseradishconjugatedantibodysolution,addwashbuffer(10mMTrispH7.5,100mMNaCl,0.1%Tween20),andwashfor30minuteswithagitation,changingthewashbufferevery3-5minutes.
- Decantwashbufferandplacetheblotinaplasticbagorcleantraycontainingchemiluminescentworkingsolution(0.125ml/cm2).Rotatethebagortraytoallowthesolutiontocoverthesurfaceofthemembranefor1-5minutes.
- Removeblotfromthebagortrayandplaceitbetweentwopiecesofwrite-onacetatetransparencyfilm.Smoothovercoveredblottoremoveairbubblesandexcesssubstrate.
EXPOSETOX-RAYFILMORANYSENSITIVESCREEN.ANINITIALEXPOSUREOF10-60SECONDSISRECOMMENDEDFORFILM.
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