western blotting

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1、AgriseraWesternBlotrecommendationsandtroubleshootingEachantibody-antigeninteractionhasuniquecharacteristics.Aprotocolgivinggoodresultsforoneantibody-antigenpairmightfoundtobeunsatisfactoryforasecondantibodyevenonthesamesample.TheinteractionofantiserawithproteinepitopesinWesternblotdetect

2、ionisdependentonanumberoffactorsallcontributingtothefinalsignal/noiseratio.Knowledgeaboutandcontrolofthosefactorsallowsformodificationsandoptimizationoftheprocedure.Polyclonalantibodies(serumorIgY-fractionsfromeggyolk)usuallycontainanumberofdifferentantibodiesinteractingindependentlywith

3、differentepitopesonthetargetusedforimmunization.Thisnumberislimitedforpeptide-antigensbutifrecombinantornativeproteinshavebeenusedforimmunizationthesignalobtainedhastoberegardedascumulative(=causedbyseveraldifferenttypesofantibodiespresentintheserum/IgYused).Toachievereproducibleresultst

4、heusageofgloves,forceps,cleananddetergent-freelaboratorymaterials,non-expiredreagents,freshlypreparedbufferswithcontrolledpH,aswellaskeepingprotocolvolumes,dilutions,andtimesisofprimaryimportanceinadditiontothestepsdiscussedbelow.Ingeneralincubationtimesshouldbeasshortaspossiblewhileanti

5、bodydilutionsshouldbeashighaspossible.(1)sample Carefulcontrolaboutthepresenceofthetargetproteininthestartingmaterial,itspreparationandstorageundernon-degrading/aggregatingconditionsisessential.Ifthetargetproteinisdegradedornotpresentinsufficientamountsinthesampleloadeditmightbeimpossibl

6、etodetect.Epitopeabundancecanbeenhancedunderalteredgrowthconditions,byselectivetissuepreparation,orfractionationsofcomplexcellularextracts(i.e.organellepreparation). Runsamplebuffer(+loadingbuffer)inonelaneofyourgeltocheckforcontributionofyoursampleextractionbuffertothebackgroundsignal.A

7、lternativepreparationmethodsshouldbeconsideredifpossible.Includepositiveandnegativecontrolsfromthebeginning:comparisonofsamplespreparedfrommaterialcontainingnotarget(e.g.genetic0-mutants)orhigheramountsofthetarget(e.g.over-expressionmutants)aswellascellularfractionation(e