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来自 : 蚂蚁淘
ToomanybandsonaWesternblot Western-orimmunoblottingisacommonlyemployedtechniqueforthedetectionofproteinantigensincomplexmixtures.SamplesarefirstseparatedbySDS-polyacrylamidegelelectrophoresis.Theseparatedproteinsarethentransferredtoamembrane(usuallynitrocellulose,polyvinylpyrolidon,ornylon).Thesemembranesareincubatedwithanantibodyspecificfortheproteinofinterestwhichbindstotheproteinbandimmobilizedonthemembrane.TheantibodyisthenvisualizedwithadetectionsystemthatisusuallybasedonasecondaryproteinbindingtoIgchainswhichislinkedtoacolor-yieldingreaction. Itisnotuncommonthat,contrarytothetheoreticalpredictions,severalbandsaredetected.AlthoughitispossIBLethattheantibodyisnotentirelyspecificfortheprotein,otherfactorsmayberesponsible: - Proteolyticbreakdownoftheantigen.Thisisnotuncommon,particularlyifsamplesarestoredforprolongedtimeorifproteinsormembranesarefractionatedafterhomogenizationofthestartingtissue.Alladditionalbandsareoflowerapparentmolecularmassthanthefull-lengthprotein.Particularlysusceptiblearesynapsinsandsynaptotagmins.AdditionofproteaseinhibitorssuchasPMSF,pepstatinorleupeptinshouldbeconsidered.
- Toomuchproteinperlaneordetectionsystemtoosensitive.OverloADIngofthegelisoneofthemostcommonreasonsfor"ghostbands".ImmobilizedproteinsmayprovideaconcentratedadsorbtivesurfacetowhichcertainIgGmaybindnonspecifically.Similarly,suchnonspecificbindingmaybeuncoveredwhenhighlysensitivedetectionsystemssuchasenhancedchemoluminescenceareemployed.Adilutionseriesofthestartingmaterialusuallyclarifieswhichofthesignalsareartefactual.
- Ineffecientblocking.Avarietyofdifferentblockingagentsaredescribedintheliteratureincludingnonionicdetergentsand/orproteins.Changeoftheblockingconditionsmayremedytheproblem.
- Concentrationofantigentoolow.TheresolutionofSDS-PAGEislimitedto50-100bands.Iftherelativeconcentrationoftheantigenofinterestistoolow(lessthan0.2%oftotalprotein),itmaybedifficulttodetect(forinstance,synaptobrevin/VAMPcomigrateswithhistonesincellhomogenateswhichinterferewithitsdetection).Signalenhancementmaythenleadtotheappearanceofartificialbands.Enrichmentoftheantigenbyfractionationorbyimmunoprecipitationshouldbeconsidered.
Allourantibodiesweretestedbyimmunoblotting.Theyrecognizesinglebandsinrelativelycrudefractions. Apartfromthis,severalbandsarenotalwaystheresultofsuboptimalexperimentalconditionsorcrossreactivity.Manyproteinshaveseveralisoforms,andanantibodymaydetectmorethanoneofthem. | |
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