1.PerformSDS-polyacrylamidegelelectrophoresis(SDS-PAGE)onacellsamplepreparedusingmodifiedRIPAbufferandtransfertheproteinstoanitrocellulosemembrane.

2.WashthenitrocellulosetwicewithdistilledH₂O.StainthenitrocellulosewithPonceauRedsolutionfor5minutestovisualproteinbands(Stocksolution:2%PonceauSin30%trichloroaceticacidand30%sulfosalicylicacid;dilute1:10foruse).RinsethemembraneinH₂OuntilproteinbandsaredistinctandmarkthepositionofthemolecularweightMarkerswithaballpointpenorpencil.ThePonceauRedstainwillbewashedoffthemembraneduringtheblockingstep.

[Note:Donotlettheblotdryoutatanystepthroughdevelopmentasthiswillcauseanincreaseinbackgroundstaining.]

3.BlocktheblottednitrocellulosemembraneinfreshlypreparedPBScontaining3%nonfatdrymilkfor20minutesatroomtemperaturewithconstantagitation.Amaximumblockingtimeof2hoursatroomtemperatureshouldnotbeexceededsincestainingartifactmayappear.Forlongerblockingtimes,themembraneshouldbekeptat4¡C.

4.Dilutetheprimaryantibodytotherecommendedconcentration/dilutioninfreshPBS/3%nonfatdrymilk(pleaseseeCertificateofAnalysisfordetailedinformation).Placethenitrocellulosemembraneintheprimaryantibodysolutionandincubate1to2hoursatroomtemperatureorovernightat4¡Cwithagitation.

5.Washthenitrocellulosemembranefivetimesfor3to5minuteseachwitheitherwaterorPBScontaining0.05%Tween20.

6.Incubatethenitrocellulosemembraneinthesecondaryreagentofchoicefor30minutesto1houratroomtemperatureorovernightat4¡Cwithagitation.Foramousemonoclonalantibody,agoat-anti-mouseHRPconjugatedantibodyisrecommended,forarabbitpolyclonalantibody,agoat-anti-rabbitHRPconjugatedantibodyisadvisable.

7.Washthenitrocellulosemembranefivetimesfor3to5minuteseachwitheitherwaterorPBScontaining0.05%Tween20.Ifthemembranehasbeenwashedwithwater,afinalwashstepfor3to5minutesinPBScontaining0.05%Tween20shouldfollow.

8.Rinsethenitrocellulosebrieflyin4to5changesofwater,thenperformdetectionofproteinsusingadetectionsystemofchoice,e.g.,enhancedchemiluminescence(ECL).

TIP:Ifitdoesnotinhibitantibodybinding,Tween20(0.05%finalconcentration)canbeaddedtotheblockingandantibodysolutionsaswelltoreducenonspecificbackgroundstaining.

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