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1、AgriseraWesternBlotrecommendationsandtroubleshootingEachantibody-antigeninteractionhasuniquecharacteristics.Aprotocolgivinggoodresultsforoneantibody-antigenpairmightfoundtobeunsatisfactoryforasecondantibodyevenonthesamesample.TheinteractionofantiserawithproteinepitopesinWesternbl
2、otdetectionisdependentonanumberoffactorsallcontributingtothefinalsignal/noiseratio.Knowledgeaboutandcontrolofthosefactorsallowsformodificationsandoptimizationoftheprocedure.Polyclonalantibodies(serumorIgY-fractionsfromeggyolk)usuallycontainanumberofdifferentantibodiesinteractingi
3、ndependentlywithdifferentepitopesonthetargetusedforimmunization.Thisnumberislimitedforpeptide-antigensbutifrecombinantornativeproteinshavebeenusedforimmunizationthesignalobtainedhastoberegardedascumulative(=causedbyseveraldifferenttypesofantibodiespresentintheserum/IgYused).Toach
4、ievereproducibleresultstheusageofgloves,forceps,cleananddetergent-freelaboratorymaterials,non-expiredreagents,freshlypreparedbufferswithcontrolledpH,aswellaskeepingprotocolvolumes,dilutions,andtimesisofprimaryimportanceinadditiontothestepsdiscussedbelow.Ingeneralincubationtimessh
5、ouldbeasshortaspossiblewhileantibodydilutionsshouldbeashighaspossible.(1)sample?Carefulcontrolaboutthepresenceofthetargetproteininthestartingmaterial,itspreparationandstorageundernon-degrading/aggregatingconditionsisessential.Ifthetargetproteinisdegradedornotpresentinsufficientam
6、ountsinthesampleloadeditmightbeimpossibletodetect.Epitopeabundancecanbeenhancedunderalteredgrowthconditions,byselectivetissuepreparation,orfractionationsofcomplexcellularextracts(i.e.organellepreparation).?Runsamplebuffer(+loadingbuffer)inonelaneofyourgeltocheckforcontributionofy
7、oursampleextractionbuffertothebackgroundsignal.Alternativepreparationmethodsshouldbeconsideredifpossible.Includepositiveandnegativecontrolsfromthebeginning:comparisonofsamplespreparedfrommaterialcontainingnotarget(e.g.genetic0-mutants)orhigheramountsofthetarget(e.g.over-expressio
8、nmutants)aswellascellularfractionation(e
