ForProteinConcentrationDeterminationofCellCulture

1. Decantmediumfrom10cmdishofadherentcellsandrinseplaterapidlywithphosphate-bufferedsaline(PBS).
2. AspirateexcessPBS.
3. Add1mlboilinglysisbuffer(1%SDS,1.0mMsodiumortho-vanadate,10mMTrispH7.4).
4. Scrapecellsfromdish,transfertoamicrocentrifugetube,andboilforanadditional5minutes.Toreduceviscosity,thesamplemaybesonicatedbrieflyorpassedseveraltimesthrougha26-gaugeneedle.
5. Centrifugethesamplefor5minutestopelletinsolublematerial,thendiscardpellet.
6. Diluteanaliquotofthecelllysatesampleatleast10-foldfortheBCA(Pierce)proteinconcentrationassay(SDSconcentrationmustbebelow0.1%toavoidinterferencewiththecolorimetricreADIng).

ForProteinGelElectrophoresisofCellCulture(withoutdeterminingproteinconcentration)

1. Decantmediumfrom10cmdishofadherentcellsandrinseplaterapidlywithphosphate-bufferedsaline(PBS).
2. AspirateexcessPBS.
3. Add1mlboiling2Xconcentratedelectrophoresissamplebuffer(125mMTrispH6.8,4%SDS,10%glycerol,0.006%bromophenolblue,1.8%beta-mercaptoethanol).
4. Scrapecellsfromdish,transfertoamicrocentrifugetube,andboilforanadditional5minutes.Toreduceviscosity,thesamplemaybesonicatedbrieflyorpassedseveraltimesthrougha26-gaugeneedle.Centrifugethesamplefor10minutestopelletinsolublematerial.Discardpellet.
5. Thecelllysatesample(supernatant)isnowreadyforloadingontoyourgel.

ForProteinConcentrationDeterminationofWholeTissue

1. Rapidlyhomogenizeevery0.25gtissuein3.5mlofboilinglysisbuffer(1%SDS,1.0mMsodiumortho-vanadate,10mMTrispH7.4).
2. Microwavefor10-15seconds.
3. Centrifugethehomogenate(16,000xg,15C)for5minutestopelletinsolublematerial,thendiscardpellet.
4. Diluteanaliquotofthetissuelysatesampleatleast10-foldfortheBCA(Pierce)proteinconcentrationassay.

Guidelinesforchoosingthepercentgeltobeusedforcertainmolecularweightproteins(basedon37:1acralyamide:bisacraylamideratio)

4-5%gels:>250kDa

7.5%gels:250-120kDa

10%gels:120-40kDa

13%gels:40-15kDa

15%gels:<20kDa

GelElectrophoresis

1. Ifnotalreadyinelectrophoresissamplebuffer,addanequalvolumeof2Xsamplebuffer(125mMTrispH6.8,4%SDS,10%glycerol,0.006%bromophenolblue,1.8%b-mercaptoethanol)toallsamplesandboilfor3-5minutes.
2. Apply5-20µgtotalproteinofcellortissuelysatetoeachwellofa0.75-1.0mmthickgel.Forthickergels(1.5mmthick),applyupto25-40µgineachwell.
3. Electrophoreseuntilthebromophenolblueinthesamplesreachesthebottomofthegel.Turnoffpowersupply.Keepgelsinrunningbufferuntilreadytotransfer.

WetTransfer

Note:Sinceextranegativechargesareneededtoreach1Ampinawettransfersystem,adjustthepHofthetransferbuffertoapproximatelypH8.0usingNaOH.

1. Fortransferofproteinssmallerthan20kDa,transferproteinsfromgeltoPVDF(polyvinylidenedifluoride)membraneat1Ampconstantcurrentfor45minsorequivalent(250mAmpfor3hoursor500mAmpfor90minutes)intransferbuffer(25mMTris,190mMglycine,20%MeOH).
2. Fortransferofproteinssmallerthan120kDa,transferproteinsfromgeltoPVDFmembraneat1Ampconstantcurrentfor1hourorequivalent(250mAmpfor4hoursor500mAmpfor2hours)intransferbuffer(25mMTris,190mMglycine,20%MeOH).
3. Forproteinslargerthan120kDa,transfertoPVDFmembraneat1Ampconstantcurrentfor90minutesorequivalent(250mAmpfor6hoursor500mAmpfor3hours)intransferbuffer+SDS(25mMTris,190mMglycine,20%MeOH,0.05%SDS).
4. 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

1. Removetheblotfromthetransferapparatusorstainingtrayandimmediatelyplaceintoblockingbuffer(1%BSA,10mMTrispH7.5,100mMNaCl,0.1%Tween20).
2. Incubatetheblotfor30minutesat37°C,1houratroomtemperature,orovernightat4°C.

1. DilutetheAPconjugatedantibodyinthecorrespondingblockingbuffer.
2. Decanttheblockingbufferfromtheblot,addtheantibodysolution,andincubatewithagitationfor30minutesat37°C,onehouratroomtemperature,orovernightat4°C.

PrepareBCIP(bromochloroindolylphosphate)bydissolving0.5gramsofthedisodiumsaltin10mlof100%dimethylformamide.PrepareNBT(nitrobluetetrazolium)inaglassvialbydissolving0.5gramsin10ml70%dimethylformamide.Storeseparatelyat4°Cindarkcontainers.

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