資源描述：
《western blotting》由會(huì)員上傳分享，免費(fèi)在線閱讀，更多相關(guān)內(nèi)容在行業(yè)資料-天天文庫(kù)。
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
