1. Preparereducedandnon-reducedsamples/controlsandseparateproteinsusingaSDS-PAGEgel.UseappropriatepercentageSDS-PAGEgelforproteinofinterest.Typically12%acrylamidegelsareusedforhighmolecularweight(MW)proteins(>50kDa),15%gelsformidrangeMWproteins(15-50kDa)and20%gelsforlowMWproteins(<15kDa).Usepre-stainedMWstandardsasanindicationofasuccessfultransfer.
2. Removestackinggelandsidesofrunninggelbeyondsamplewellswitharazorblade.Notchbottomright-handcornerofgelfororientation.Note:EquilibrationofrunninggelinCathodeBufferisgenerallynotrequiredbutmayimproveresultsifbandsappeardiffuse.
3. PreparetransfermembranebysoakinginWettingSolutionforafewseconds.EquilibratemembraneinAnodeBufferIIfor5minutes.
4. WettwopiecesoffilterpaperinAnodeBufferIandplaceonanodeplateofblotter.Avoidtrappingairbetweenelectrodeandfilterpaperbylayingfilterpaperonelectrodesatanobliqueangle.
5. WetonepieceoffilterpaperinAnodeBufferIIandplaceontopoffilterpaperspreviouslyplacedonelectrode.
6. RemovetransfermembranefromAnodeBufferIIandplaceontopoffilterpaperstack.
7. Placegelontopoftransfermembranetakingcarenottotrapairbubblesbetweengelandmembrane.
8. WetthreepiecesoffilterpaperinCathodeBufferandplaceontopofgel.Useacleanplastictesttubetorolloutairbubbles.
9. Placecathodeplateofblotterontopoftransferstack(seeFigure1).
10. Connecthighvoltagecordstopowersupply.Applyaconstantcurrentof1.9-2.5mApercm2ofgelareafor30-60minutes.Transfertimedependsuponproteinsbeingtransferred.
11. Aftertransferiscomplete,turnoffpowersupplyandremovecathodeplateofblotter.Removetransfermembraneandcutlowerrightcornerofmembranetomarkorientationofgel.
12. Ifimmunostainingistobeperformedimmediately,rinsethemembraneindistilledwaterandproceedtotheimmunostainingportionofprotocol.Ifstainingistobeperformedatalatertime,placemembraneonapapertowelandallowtodry.Storemembraneinadrycontainerat4°C.

WesternBlotProtocolforCellLysates

ThiswesternblottingprotocolisintendedtoprovideadditionaldetailfortheuseofR&DSystemsantibodiesinwesternblotting.PleaseconsulttheproductinsertfortheappropriateconcentrationofprimaryantibodyandthecompositionofWashBuffer,BlockingSolution,andBlottingBuffer.Topreparetotalcelllysates,addenoughPBStocellpellettomakethefinalconcentration3x106cells/ml.Thisisaconvenientcelldensityformanycelllines,butadjustmentsmaybenecessaryforcelltypesthatdiffersubstantiallyinsizeandproteincontent.Preparecellextractsinappropriatenon-reducingorreducingsamplebufferasindicatedontheproductinsert.Insomecasesreducingagentscandestroytheepitopethatisrecognizedbyamonoclonaldetectionantibody.MixcellsUSPensionwithanequalvolumeofnon-reducing2XSDSgelsamplebuffer(6%SDS,0.25MTris,pH6.8,10%glycerol,andbromophenylblue)orreducing2XSDSgelsamplebuffer(non-reducingbufferplus20mMdithithreitol).SonicatecellstofragmentDNAusing8-10burstsof2-3secondseach.

1. LoadcellextractsandseparateproteinsonanSDSpolyacrylamidegel.
2. TransfertheelectrophoresedproteinsontoanImmobilonPmembrane(Millipore)andincubatethemembranefor1houratroomtemperatureorovernightat2-8°CinBlockingSolution.
3. Washthemembraneatroomtemperaturefor30minuteswith3ormorechangesofWashBuffer.
4. Incubatethemembranefor2hoursatroomtemperatureorovernightat2-8°CinBlottingBuffercontainingprimaryantibody.
5. Washthemembraneatroomtemperaturefor30minuteswith3ormorechangesofWashBuffer.
6. Incubatethemembraneatroomtemperaturefor1hourinBlottingBuffercontaininganappropriatesecondaryreagentsuchasgoatanti-mouse-HRPat1:4000(Catalog#HAF007).
7. Washthemembraneatroomtemperaturefor30minuteswith3ormorechangesofWashBuffer.
8. Detectwithchemiluminescencereagents.
9. Optimaldilutionsshouldbedeterminedbyeachlaboratoryforeachapplication.