TroubleshootingGuide:WesternBlot
Problem:PoorTransfer
PossIBLeSource TestorAction Membranedoesnotwetuniformly Pre-wetmembranein100%methanol MWofproteinislessthan10,000 IfproteinhasMW<10,000,itmayhave揵lownthrough?themembrane.Reducetransfertime,orplacetwosheetsoftransfermembranesoakedinAnodeBufferIIundergelintransferstack,orselectmembranewithsmallerporesize Isoelectricpointofproteinis<9 Usealternatebuffersystem(suchasCAPSbufferpH10.5)withahigherpHAssembletransferstackwithadditionalmembranesoakedincathodebufferonthecathodesideofgel.Thiswillcaptureproteinsthatmigratetowardsthecathode.Identifymembranelocationinstackandimmunostainallmembranesinstack Methanolconcentration Highermethanolconcentrationincreasesbindingtomembranebutmayretardtransferfromgel SDSconcentration Adding0.005-0.01%SDStocathodebuffercanincreasetransferefficiencyofproteinfromgelNote:IncreasedSDSconcentrationmayinterferewithbindingtomembrane Thickgel ThickergelsorhigherMWproteinsmayrequirelongertransfertimes Problem:HighBackgroundStaining
PossibleSource TestorAction Membranedoesnotwetuniformly Pre-wetmembranein100%methanol Inadequateblocking Optimizetheblockingstepbytryingalternateblockingsolutions(e.g.,non-fatmilk,gelatin,etc.)orincreasingtimeandtemperatureofblockingstep Cross-reactivityofantibodyreagents Checkforcross-reactivityofantibodyreagentstotheblockingprotein Problem:LowProteinBinding
PossibleSource TestorAction Overwashing KeepthelengthofTTBSwashestoaminimumSee揚oorTransfer?problemforalternatives Problem:HighProteinBinding,LowSignal
PossibleSource TestorAction Inadequateantibodystaining Checkantibodydilutionsandexpirationdates InactiveAPconjugateand/orsubstrate Addenzymeconjugatetosubstratereagentsaspreparedinstep7ofimmunostainingprocedure.Ifcoloroccurs,reagentsareperformingproperly Poorsample Ifpositivecontrolworked,samplemaynotcontainproteinofinterestoritmaybepresentatconcentrationstoolowtodetect Problem:SeveralBandsareStained
PossibleSource TestorAction Non-specificbindingofsecondaryreagents Checknon-specificbindingbyrunningextrasamplelaneandomitting1?antibodyfromimmunostainingprocedure,runwithanon-specificantibodycontrolinplaceof1?antibody,orperformdotblotandomitthe1?antibody.Ifstainingoccurs,chooseanalternate2?antibody Primaryantibodyisnotmonospecific Useamonoclonaloraffinitypurifiedantibody