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.