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

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