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首页 蛋白纯化策略

蛋白纯化策略.pdf

蛋白纯化策略

doggystone
2013-07-17 0人阅读 举报 0 0 暂无简介

简介:本文档为《蛋白纯化策略pdf》,可适用于工程科技领域

GEDevelopinganefficientproteinpurificationschemeGEIntroductionThreephasestrategyCombiningtechniquesPurityrequirementsCharacteristicsofthetargetproteinandcontaminantsExamplesSummaryandshortcutsDevelopinganefficientproteinpurificationschemeDevelopinganefficientproteinpurificationschemeGEProteinPurificationAimsProteinPurificationAimsSufficientpurityandquantityMaintainedbiologicalactivityGoodeconomyGEYieldsfromMultistepProteinPurificationsYieldsfromMultistepProteinPurificationsNumberofstepsYield()stepstepstepstepstepGEInputforPurificationProtocolDevelopmentInputforPurificationProtocolDevelopmentThreephasestrategyPurificationprotocolRequiredpurityandquantityPhysicalchemicalpropertiesoftargetandmaincontaminantsSourcematerialinformationSeparationtechniqueknowledgeScoutingrunsandoptimizationEconomyandresourcesGEProteinPurificationProteinPurificationAnalyticaltools•Arapidandreliableassayforthetargetprotein•Puritydetermination(egSDSPAGE)•Totalproteindetermination(egcolorimetricmethod)GEThreePhaseStrategyThreePhaseStrategyPurityStepCaptureIntermediatepurificationPolishingIsolateproduct,concentrate,stabilizeRemovebulkimpuritiesAchievefinalpurityRemovetraceimpurities,structuralvariants,aggregatesetcGECaptureCaptureResolutionSpeedRecoveryCapacity‹Initialpurificationofthetargetmoleculefromcrudeorclarifiedsourcematerial‹Concentrationandstabilization(egremovalofproteases)GEIntermediatePurificationIntermediatePurificationResolutionSpeedRecoveryCapacity‹RemovalofbulkimpuritiesGEPolishingPolishingResolutionSpeedRecoveryCapacity‹Finalremovaloftracecontaminants,egstructuralvariantsofthetargetproteinGEThreePhaseStrategyRankingofChromatographyTechniquesThreePhaseStrategyRankingofChromatographyTechniquesTechniqueCaptureIntermediatePolishingGFIEXHICACRPCConsiderationsLimitedsamplevolumeLimitedflowraterangeProteinligandissensitivetoharshcleaningconditionsUseoforganicsolvents,lossofbiologicalactivityGELinkingChromatographyTechniquesintoaPurificationProtocolGeneralRulesLinkingChromatographyTechniquesintoaPurificationProtocolGeneralRulesCombinetechniqueswithcomplementaryselectivities(egIEX,HICandGF)Minimizesamplehandlingbetweenpurificationsteps(egconcentration,bufferexchange)GELinkingChromatographyTechniquesLinkingChromatographyTechniquesTechniqueEndconditionsStartconditionsSmallsamplevolumeGFDilutedsampleBufferchange(ifrequired)LowionicstrengthIEXHighionicstrengthorpHchangeHighionicstrengthHICLowionicstrengthSpecificbindingconditionsACSpecificelutionconditionsGELinkingChromatographyTechniquesLinkingChromatographyTechniquesIEXHICGFACGFRPCIEXHICGFACGF(NH)SOHICIEXGFHICGFIEXGFGF(desalting)ACGFGEPurityRequirementsPurityRequirements‹Contaminantswhichdegradeorinactivatethetargetprotein(egproteases),needtobereducedto“nondetectable”levels‹Contaminantswhichinterferewithsubsequentanalysesneedtobereducedto“nondetectable”levels‹Itisbetterto“overpurify”thanto“underpurify”GE•Therapy•Invivostudies•Crystallizationforxraystudies•Nterminalsequencingofanunknownprotein•Mostphysicalchemicalcharacterizationmethods•AntigenformonoclonalantibodyproductionExtremelyhighHighModeratePurityRequirementsBriefGuidelinesPurityRequirementsBriefGuidelinesGETowardstheOptimalPurificationProtocolAccountingforTargetProteinProperties()TowardstheOptimalPurificationProtocolTowardstheOptimalPurificationProtocolAccountingforTargetProteinProperties()AccountingforTargetProteinProperties()TargetproteinpropertyPurificationparameteraffected‹StabilitywindowzpHzIonicstrengthzCofactorszDetergentconcentrationzOrganicsolventszOther(light,oxygenetc)IEXconditions(alsoACandRPC)HICconditionsselectionofbuffers,pH,salts,additivesbufferadditivesRPCconditionsvariousGETowardstheOptimalPurificationProtocolAccountingforTargetProteinProperties()TowardstheOptimalPurificationProtocolTowardstheOptimalPurificationProtocolAccountingforTargetProteinProperties()AccountingforTargetProteinProperties()Physicalchemicalproperties•Chargeproperties(isoelectricpoint)•Molecularweight•Posttranslationalmodifications•BiospecificaffinityTargetproteinpropertyPurificationparameteraffectedselectionofIEXconditionsselectionofGFmediumselectionofgroupspecificACmediumselectionofligandforACGETargetProteinStabilityWindowTargetProteinStabilityWindowDeterminationofasuitableammoniumsulfateconcentrationandpHscreeningrangeforHICGETargetProteinPropertiesSelectionofionexchangeconditionsTargetProteinPropertiesSelectionofionexchangeconditionspHmoleculeschargeElectrophoretictitrationcurveofchickenbreastmuscleusingzymogramdetectionforcreatinekinaseTargetproteinContaminantsGEGProteinReceptorKinasePurificationGProteinReceptorKinasePurificationTechniquePptHICAIEXCIEXACPurificationfactorComment•Allbufferscontainproteaseinhibitors•AllpurificationsdoneatoC•Removalstep,maincontaminantisbound•Elutionbufferisusedasstartingbufferfornextcolumn•µghomogenousproteinobtainedATobinetal()JBiolChem,PorcinecerebellahomogenateRESOURCEQAmmoniumsulfateprecipitationButylSepharoseFastFlowRESOURCEsHiTrapHeparinGERecαMannosidasePurificationfromPichiaRecαMannosidasePurificationfromPichiaTechniquePurificationfactorComment•µghomogenousproteinobtainedYFLiaoetal()JBiolChem,•CapturewithstepgradientmgoftotalproteinappliedUFGFAIEXHICPhenylSepharoseHPQSepharoseFFSuperdexpgUltrafiltrationGEDNABindingProteinPurificationDNABindingProteinPurificationTechniqueDNASepharoseCIEXACACACCIEXPurificationfactorComment•GeneralACstepforDNAbindingproteins•Removalstep,nonspecificDNAbindingactivityremoved•Mainpurificationstep•Finalpolishing,µgproteinobtainedJBerthelsenetal()JBiolChem,•RapidcaptureHeLacellnuclearextractsSPSepharoseHighPerformanceHeparinSepharoseFastFlowDNASepharoseMonoSGE•Finalpolishingandpuritycheck,µgobtainedMembraneProteinPurificationMembraneProteinPurificationTechniqueACAIEXCIEXACCIEXPurificationfactorComment•Negativestepcontaminantremoved•Detergentexchange,volumereductionbeforeAC•MainpurificationstepTWhiteetal()JBiolChem,•Stepgradient,rapidconcentratingcapturestepPlacentaextractinTritonXBlueSepharoseDEAESephacelSPSepharoseFFMucSepharoseMonoSGETowardsaGeneralProteinPurificationProtocolTowardsaGeneralProteinPurificationProtocolArapidmethodforobtainingmilligramquantitiesofdifferentrecombinantproteins,forinitialcharacterizationstudiesSemiautomatedinÄKTAexplorer,withpremademethodtemplatesandBufferPrepIonexchangeSTREAMLINESPorDEAESPorQSepharoseFFHydrophobicinteractionPhenylSepharoseFF(highsub)GelfiltrationSuperdexprepgradeGETowardsaGeneralProteinPurificationProtocolResultswithEcolirProteinsTowardsaGeneralProteinPurificationProtocolResultswithEcolirProteinsIonexchangeSTREAMLINESPorDEAESPorQSepharoseFFHydrophobicinteractionPhenylSepharoseFF(highsub)GelfiltrationSuperdexprepgradeProteinExpressionCapturestep(purifiedtohomogeneity)AnnexinVExtracellularSTREAMLINEDEAEαAmylaseIntracellularSTREAMLINEDEAEantigpFabPeriplasmicSPSepharoseFastFlowGEShortcutsRapidEstablishmentofMilligramScalePurificationProtocolsShortcutsRapidEstablishmentofMilligramScalePurificationProtocolsIfabiospecificligandisavailable:useACasthemainpurificationstepIfthepurificationisnotintendedtobescaledup:usehighperformancemedia(egMonoBeads)throughoutFor“oneofakind”purificationofaproteinegforsequencingbeforegeneisolation:sacrificeyieldforpuritybymakingnarrowcutsIfnothingisknownabouttargetproteinandcontaminantsproperties:trytheIEXHICGFcombinationEstablishafastandreliableassayforthetargetproteinGEASystematicApproachtoPurificationDevelopmentSummaryASystematicApproachtoPurificationDevelopmentSummaryDevelopassaymethodsSettheaims(purityandquantity)CharacterizethetargetproteinUsedifferentseparationprinciplesUsefewstepsLimitsamplehandlingbetweenpurificationstepsStartwithhighselectivityincreaseefficiencyRemoveproteasesquicklyReducevolumeinearlystepKeepitsimple!

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