TECHTIP#22PierceBiotechnologyPOBox117(815)968-0747www.thermo.com/pierce3747N.MeridianRoadRockford,lL61105USA(815)968-7316faxIntroductionWesternblottingproceduresdependonappropriateinteractionsamongmultiplecomponents,includingmembrane,antigen,blockingbuffer,primaryandsecondaryantibodies,andsubstrate.Inaparticularexperiment,anyoneoftheseinteractionscancontributetoexcessivebackgroundsignalinWesternblottingexperiments.ThisTechTipdescribesseveraldifferentdotblotproceduresthatcanbeusedtodeterminethesourceofbackgroundcausedbynonspecificinteractionsamongsystemcomponentsinaWesternblottingprocedurethatusesachemiluminescentsubstrateforhorseradishperoxidase,suchasThermoScientificSuperSignalorPierce®ECLSubstrates.BeforeconcludingthatnonspecificbackgroundiscausedbyanincompatibilitybetweenWesternblotcomponentsandusingtheproceduredescribedhere,makesurethatthecorrectprimaryandsecondaryantibodydilutionshavebeenusedintheoriginalprocedure.SuperSignalChemiluminescentSubstratesareverysensitive,andusinginappropriateantibodydilutionscanresultinavarietyofunexpectedresults:•Weaksignal•Signalfadesquickly•Nosignalwithnobackground•Highbackgroundwithwhitebands(reverseimage)•Orangeorbrownspotsonthemembrane•Nonspecificbands•Diffusebands•BlotchyorspeckledbackgroundConsideroptimizingprimaryandHRP-conjugatedsecondaryantibodydilutions(seeTechTip#24)beforeorinadditiontotestingfornonspecificbackgroundsignal.MaterialsRequired•Nitrocellulosemembranecutinto1cmx8cmstripsthatmaybetreateddifferentlytodeterminesourcebackground•Antigen-containingsample,dilutedinphosphatebufferedsaline(PBS),Trisbufferedsaline(TBS)orsimilarbuffer•PrimaryandHRP-conjugatedsecondaryantibodies•BlockingBuffer:testtheblockingbufferthatresultedinhighbackgroundintheoriginalprocedure•WashBuffer,eitherPBSorTBS,withTween®-20addedtoafinalconcentrationof0.05%•Substrateworkingsolution(WS):chemiluminescentSubstrateforHRP(e.g.,SuperSignalSubstrate)•FilmorCCDcameraapparatustodetectchemiluminescenceProcedureCutapieceofmembranei...
