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ID:16038923
大小:262.50 KB
页数:8页
时间:2018-08-07
《western blotting》由会员上传分享,免费在线阅读,更多相关内容在行业资料-天天文库。
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
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