western blotting

1、AgriseraWesternBlotrecommendationstroubleshootingEachantibodyantigeninteractionhasuniqueacteristics.Aprotocolgivinggoodresultsfoneantibodyantigenpairmightfoundtobeunsatisfactyfasecondantibodyevenonthesamesample.Theintera

2、ctionofantiserawithproteinepitopesinWesternblotdetectionisdependentonanumberoffactsallcontributingtothefinalsignalnoiseratio.Knowledgeaboutcontrolofthosefactsallowsfmodificationsoptimizationoftheprocedure.Polyclonalantib

3、odies(serumIgYfractionsfromeggyolk)usuallycontainanumberofdifferentantibodiesinteractingindependentlywithdifferentepitopesonthetargetusedfimmunization.Thisnumberislimitedfpeptideantigensbutifrecombinantnativeproteinshave

4、beenusedfimmunizationthesignalobtainedhastoberegardedascumulative(=causedbyseveraldifferenttypesofantibodiespresentintheserumIgYused).Toachievereproducibleresultstheusageofglovesfcepscleandetergentfreelabatymaterialsnone

5、xpiredreagentsfreshlypreparedbufferswithcontrolledpHaswellaskeepingprotocolvolumesdilutionstimesisofprimaryimptanceinadditiontothestepsdiscussedbelow.Ingeneralincubationtimesshouldbeasshtaspossiblewhileantibodydilutionss

6、houldbeashighaspossible.(1)sampleCarefulcontrolaboutthepresenceofthetargetproteininthestartingmaterialitspreparationstageundernondegradingaggregatingconditionsisessential.Ifthetargetproteinisdegradednotpresentinsufficien

7、tamountsinthesampleloadeditmightbeimpossibletodetect.Epitopeabundancecanbeenhancedunderalteredgrowthconditionsbyivetissuepreparationfractionationsofcomplexcellularextracts(i.e.ganellepreparation).Runsamplebuffer(loadingb

8、uffer)inonelaneofyourgeltocheckfcontributionofyoursampleextractionbuffertothebackgroundsignal.Alternativepreparationmethodsshouldbeconsideredifpossible.Includepositivenegativecontrolsfromthebeginning:comparisonofsamplesp

9、reparedfrommaterialcontainingnotarget(e.g.geic0mutants)higheramountsofthetarget(e.g.overexpressionmutants)aswellascellularfractionation(e.g.ganellpreparation)willallowtocontrolfspecificityofthesignalsobtained.(3)transfer

10、Theproteintransferonasuitablemembrane(blotting)withproteinbindingcapacity(nitrocellulosePVDF)ishighlydependentonthebiochemicalpropertiesofthetargetprotein.Highermoleculartargets(apparentmolecularmassesof100kDa)requirelon

11、gerblottingtimesthansmallerproteins.Atlowfieldstrengths(35Vcm)proteinsmightpassthroughthemembranewithoutbinding.Thetransfertimesshouldbeasshtaspossibletoavoidproteins(especiallylowermolecularweight)frompassingthroughthem

12、embrane.Thiscanbemonitedbyplacing2membranesontopofeachothercomparingsignalsobtainedafterincubationwiththeantibody.Ingradientgelstheposityofthegelismatchedwiththesizeoftheproteinswhichcanbeanadvantagefefficienttransfer.Ch

13、eckefficiencyofatransferbyposttransferstainingofthegel(e.g.CoomassieSilver)thefilter(Ponceaureversible).Dryingofamembrane(airdrybetweencleansheetsoffilterpaper)canimproveimmobilizationoftheprotein.Blottingequipmentshould

14、becarefullyrinsedwithdestilledwateraftereachusebeckeptawayfromcontaminatingdetergents.Foampadsshouldbecleanedwith(suitable)detergentsfollowedbythoughlywashing.Replacefoampadsiftheystarttoaquirecolours(fromplantmaterial)l