2014年2015工作文件优宁维学术

ImmunoprecipitationImmunoprecipitationEnrichand(or)purifyProtein IP，Co-IP，Pull-

Ab-Antigenin Pull-

Non-Presence,relativeabundance,size,up-regulationordown-regulation,stability,post-translationalmodificationand ctionsofPurified StepStepByStepGuidetoStepStepByStepGuidetoSamplepreparationandcellPreclearingoftheIncubationwithantibodyandformationofantibody-antigencomplexesPrecipitationofthecomplexofWashingoftheantibody-antigencomplexestoremovenon-specificproteinsand ysisofcomplexes/antigenofinterestby1.1.Samplepreparationandcell solubleproteinlysisbuffer（不带去垢剂的Non-denaturinglysisbuffer（非变性裂解液RIPA(RadioImmunoPrecipitationAssay)buffernuclearDenaturinglysisbuffer/bufferfornon-detergent（变性裂解液）(e.g.Histone，组蛋白Detergent,non-ionic(NP40,TritonX-100):0.1-2%Detergent,ionic(SDS,etc):0.01-0.5%1.1.SamplepreparationandcelltemperatureNaCl150mM（Salt：0-10%glycerol（甘油ProteaseinhibitorCation（某些特别的阳离子）ATPGTP；二价EDTA:0--5BestfreshlyStepStepByStepGuidetoSamplepreparationandcellPreclearingoftheIncubationwithantibodyandformationofantibody-antigencomplexesPrecipitationofthecomplexofWashingoftheantibody-antigencomplexestoremovenon-specificproteinsand ysisofcomplexes/antigenofinterestby2.Preclearing2.Preclearingofthe2.Preclearing2.Preclearingofthe 同时加入Protein StepStepByStepGuidetoSamplepreparationandcellPreclearingoftheIncubationwithantibodyandformationofantibody-antigencomplexesPrecipitationofthecomplexofWashingoftheantibody-antigencomplexestoremovenon-specificproteinsand ysisofcomplexes/antigenofinterestby3.3.IP:IncubationAntibodyandFormationofAntibody-AntigenComplexes100-500µg3.3.IP:IncubationAntibodyandFormationofAntibody-AntigenComplexesStepStepByStepGuidetoSamplepreparationandcellPreclearingoftheIncubationwithantibodyandformationofantibody-antigencomplexesPrecipitationofthecomplexofWashingoftheantibody-antigencomplexestoremovenon-specificproteinsand ysisofcomplexes/antigenofinterestby4.4.Precipitationofthecomplexof选择ProteinAProteinG要根据一抗的来源种属，或者选择ProteinAProteinG的混合物 StepStepByStepGuidetoSamplepreparationandcellPreclearingoftheIncubationwithantibodyandformationofantibody-antigencomplexesPrecipitationofthecomplexofWashingoftheantibody-antigencomplexestoremovenon-specificproteinsand ysisofcomplexes/antigenofinterestby5.5.Washingoftheantibody-antigenStepStepByStepGuidetoSamplepreparationandcellPreclearingoftheIncubationwithantibodyandformationofantibody-antigencomplexesPrecipitationofthecomplexofWashingoftheantibody-antigencomplexestoremovenon-specificproteinsandcontaminants ysisofcomplexes/antigenofinterestbyysisofcomplexes/antigeninterestbySDS- FactorsFactorsAffecting

Columnvs.ba FactorsFactorsAffectingMethodColumnmethodvs.batchGravityflowversusspinLargescale GravityVerysmallscale:SpinFactorsFactorsAffectingTypeofThemostprevalenttypeinresearchscaleEasytouse，durableandWithstandcentrifugationupto5000×g,pressuresof100psiandupto120°Cwithoutsignificantlossofstructureorflowrate；Lownon-specificbindingincomplexsamplesandisnotharmedbylevelsofmostdetergents,salts,mostorganicsolventsorextremesofpH(willtoleratepHrange3-14)；OtherCrosslinkedacrylamide；Ferrous(ironoxide)magneticbeads,cellulosepolystyreneparticlesor tewellsandcontrolledporeFactorsFactorsAffectingImmobilizationofAntibody(IPandCo-ProteinA,ProteinGorProteinA/GimmobilizationofProteinAbindsexclusivelytotheFcProteinGalsopreferentiallybindstoFcregion(bindlightchainsinsomeProt.Prot.Prot.Prot.HumanSSSRatWMMHumanSSSRatSSHumanWSSRatWWHumanSSSRatSSSHumanWWCowWSSHumanMMCowSSSHumanSheepWSSHumanWWSheepSSSHumanWWGoatWSSHumanWWGoatSSSHumanWWWChickenHumanWWHamsterMMMMouseWMMPigSWSMouseSSSHorseWSSMouseSSSRabbitSSSMouseSSSCatSWSMouseMonkeySSSKey:W=weakbinding,M=mediumbinding,S=strongbinding,NB=nobinding,*=RhesusForIgMantibody，useantiIgMantibodycoupledbeadsorProteinFactorsFactorsAffectingDirectimmobilizationofIPantibodydoesnotbindtotheseproteinsorwhentheantigen-containingsampleisserum(whichcontainsimmunoglobulinsthatwouldcompetewiththeIPantibodyforbinding).Covalent,directimmobilizationofIPFactorsFactorsAffectingCrosslinkingimmobilizedCrosslinker：DSSandBS3，NHSestersreactwithprimaryaminestoformcovalentamidebonds.FactorsFactorsAffectingImmobilizationofBait(Pull-IMAC/HistidinetagSuchtaghasauniqueabilitytobindtodivalentcationssuchasnickel,cobaltorGlutathione/GSTThesystemisgenerallynotconsideredasrobustasHis-tagmethodologiesbecauseGSTisanenzymeandmustremainfunctionalinordertobindtheglutathionesubstrate.Avidin/biotinWhenthebiotinylatedbaitproteinisanantibody,thebiotin-basedpull-downisactuallyaformofFactorsFactorsAffectingOrderOfProteinA/Gsupports,Antibody,ProteinHighestyieldandco-elutionof(Ab+Support)+Proteinsample;Higheryieldandpurity(Ab+support+proteinsample)together;lowestyieldandpurityFactorsFactorsAffectingSamplePre-Clearing(andReducingnon-specificBindingAntibody-antigen ctionsarefairlyBait-prey ctionsrangeinstrengthandtimefromirreversibleandlong-livedlabileandtransient(mayneedProteinAbindsIgGbestatpH8.2,while umIgGbindingoccurswithProteinGinbuffersatpH5.0.FactorsFactorsAffectingWash ction:PBSorTBS;Moderate Pluslowlevels(typically0.5-1.0%)ofNP®-40,Triton®-X-100,Strong Increasingthesodiumchloride(NaCl)concentrationto0.5Moreven1Lowlevelsofreducingagents(suchas1-2mMDTTorBME);Pull-down(Histag)lowlevelsofimidazoleorEDTA;FactorsFactorsAffectingElutionReducingbuffer:SDSsamplebuffer;Advantage：extractionmethodishighlyefficientandtheresultingsampleismoreconcentrated.Advantage：ThelowpHconditiondissociatesmostantibody-antigen ctions.Italsohelpstoweakenthein betweentheantibodyandthebeads.Andbeadscanbereusedafterremovaloftheglycinebuffer.Howevertheelutedsampleshouldbeimmedia neutralizedwithTris,pH8.0-8.5UreaBufferAdvantageousformassspectrometrybecausethesamplecanbedigestedbyproteolyticenzymes.FactorsFactorsAffectingPull-downassay:Needsystem-specificcondition(plusBiotinbinding:Need8Mguanidine•HCl(pH1.5)advantageforef