western blotting

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1、AgriseraWesternBlotrecommendationsandtroubleshootingEachantibody-antigeninteractionhasuniquecharacteristics.Aprotocolgivinggoodresultsforoneantibody-antigenpairmightfoundtobeunsatisfactoryforasecondantibodyevenonthesamesample.Theinteractionofantiserawithproteine

2、pitopesinWesternblotdetectionisdependentonanumberoffactorsallcontributingtothefinalsignal/noiseratio.Knowledgeaboutandcontrolofthosefactorsallowsformodificationsandoptimizationoftheprocedure.Polyclonalantibodies(serumorIgY-fractionsfromeggyolk)usuallycontainanum

3、berofdifferentantibodiesinteractingindependentlywithdifferentepitopesonthetargetusedforimmunization.Thisnumberislimitedforpeptide-antigensbutifrecombinantornativeproteinshavebeenusedforimmunizationthesignalobtainedhastoberegardedascumulative(=causedbyseveraldiff

4、erenttypesofantibodiespresentintheserum/IgYused).Toachievereproducibleresultstheusageofgloves,forceps,cleananddetergent-freelaboratorymaterials,non-expiredreagents,freshlypreparedbufferswithcontrolledpH,aswellaskeepingprotocolvolumes,dilutions,andtimesisofprimar

5、yimportanceinadditiontothestepsdiscussedbelow.Ingeneralincubationtimesshouldbeasshortaspossiblewhileantibodydilutionsshouldbeashighaspossible.(1)sample Carefulcontrolaboutthepresenceofthetargetproteininthestartingmaterial,itspreparationandstorageundernon-degradi

6、ng/aggregatingconditionsisessential.Ifthetargetproteinisdegradedornotpresentinsufficientamountsinthesampleloadeditmightbeimpossibletodetect.Epitopeabundancecanbeenhancedunderalteredgrowthconditions,byselectivetissuepreparation,orfractionationsofcomplexcellularex

7、tracts(i.e.organellepreparation). Runsamplebuffer(+loadingbuffer)inonelaneofyourgeltocheckforcontributionofyoursampleextractionbuffertothebackgroundsignal.Alternativepreparationmethodsshouldbeconsideredifpossible.Includepositiveandnegativecontrolsfromthebeginnin

8、g:comparisonofsamplespreparedfrommaterialcontainingnotarget(e.g.genetic0-mutants)orhigheramountsofthetarget(e.g.over-expressionmutants)aswellascellularfractionation(e