western blotting

《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.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