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首页 Gel-filtration

Gel-filtration.pdf

Gel-filtration

chenpapery
2011-08-09 0人阅读 举报 0 0 0 暂无简介

简介:本文档为《Gel-filtrationpdf》,可适用于医药卫生领域

EditionAIGelFiltrationHandbook–PrinciplesandMethodswwwchromatographyamershambiosciencescomProduction:RAKDesignABPrinciplesandMethodsGelFiltrationHandbooksfromAmershamBiosciencesÄKTA,FPLC,PlusOne,HiLoad,HiTrap,HiPrep,Hybond,ECL,ECLPlus,BioProcess,MabSelect,Sephacryl,Sephadex,Superose,Sepharose,SuperdexandDropDesignaretrademarksofAmershamBiosciencesLimitedAmershamandAmershamBiosciencesaretrademarksofAmershamplcCoomassieisatrademarkofICIplcMicroSpinisatrademarkofLidaManufacturingCorpTritonisaregisteredtrademarkofUnionCarbideChemicalsandPlasticsCoTweenisaregisteredtrademarkofICIAmericas,IncEppendorfandMultipipettearetrademarksofEppendorfNethelerHinzGmbHBIACOREisatrademarkofBiacoreABAllgoodsandservicesaresoldsubjecttothetermsandconditionsofsaleofthecompanywithintheAmershamBiosciencesgroupthatsuppliesthemAcopyofthesetermsandconditionsisavailableonrequest©AmershamBiosciencesAB–AllrightsreservedAmershamBiosciencesAB,Björkgatan,SEUppsala,SwedenAmershamBiosciencesUKLimited,AmershamPlace,LittleChalfont,BuckinghamshireHPNA,EnglandBiosciencesCorp,CentennialAvenue,POBox,PiscatawayNJ,USAAmershamBiosciencesEuropeGmbH,MunzingerStrasse,DFreiburg,GermanyAmershamBiosciencesKK,SankenBldg,HyakuninchoAmershamShinjukuku,Tokyo,JapanPercollMethodologyandApplicationsFicollPaquePlusForinvitroisolationoflymphocytesGSTGeneFusionSystemHandbookDElectrophoresisusingimmobilizedpHgradientsPrinciplesandMethodsAntibodyPurificationHandbookTheRecombinantProteinHandbookProteinAmplificationandSimplePurificationProteinPurificationHandbookIonExchangeChromatographyPrinciplesandMethodsAffinityChromatographyPrinciplesandMethodsHydrophobicInteractionChromatographyPrinciplesandMethodsGelFiltrationPrinciplesandMethodsReversedPhaseChromatographyPrinciplesandMethodsExpandedBedAdsorptionPrinciplesandMethodsChromatofocusingwithPolybufferandPBEMicrocarriercellculturePrinciplesandMethodsGelfiltrationPrinciplesandMethodsContentsIntroductionSymbolsCommonabbreviationsChapterGelfiltrationinpracticeIntroductionSeparationbygelfiltrationResolutioningelfiltrationMediaselectionSamplepreparationBuffercompositionandpreparationDenaturing(chaotropic)agentsanddetergentsColumnandmediapreparationSampleapplicationElutionandflowratesMethoddevelopmentforhighresolutionfractionationCareofgelfiltrationmediaEquipmentselectionScalingupBioProcessMediaforlargescaleproductionTroubleshootingChapterGelfiltrationmediaComponentsofgelfiltrationmediaSuperdex:firstchoiceforhighresolution,shortruntimesandhighrecoverySeparationoptionsSeparationexamplesPerformingaseparationCleaningMediacharacteristicsChemicalstabilityStorageSephacryl:fast,highrecoveryseparationsatlaboratoryandindustrialscaleSeparationoptionsSeparationexamplesPerformingaseparationCleaningMediacharacteristicsChemicalstabilityStorageSuperose:broadfractionationrange,butnotsuitableforindustrialscaleseparationsSeparationoptionsSeparationexamplesPerformingaseparationCleaningMediacharacteristicsChemicalstabilityStorageSephadex:rapidgroupseparationofhighandlowmolecularweightsubstances,suchasdesalting,bufferexchangeandsamplecleanupSeparationoptionsSeparationexamplesPerformingaseparationScaleupandprocessinglargesamplevolumesMediacharacteristicsColumnPackingCleaningChemicalstabilityStorageChapterGelfiltrationintheoryDefiningtheprocessSelectivitycurvesandmediaselectionResolutionChapterMolecularweightdeterminationandmolecularweightdistributionanalysisChapterSephadexLHSeparationoptionsSeparationexamplesPackingacolumnPerformingaseparationCleaningMediumcharacteristicsChemicalstabilityStorageTransferringSephadexLHbetweenorganicsolventsChapterGelfiltrationinaPurificationStrategy(CIPP)ApplyingCIPPSelectionandcombinationofpurificationtechniquesGelfiltrationasapolishingstepAppendixColumnpackingandpreparationColumnsforpackinggelfiltrationmediaCheckingcolumnefficiencyColumnpackingforhighresolutionfractionationusingSuperdexprepgradeandSephacrylHighResolutionColumnpackingforgroupseparationsusingSephadexAppendixSephadexandDarcy'slawAppendixSamplepreparationSamplestabilitySampleclarificationSpecificsamplepreparationstepsRemovaloflipoproteinsRemovalofphenolredRemovaloflowmolecularweightcontaminantsAppendixSelectionofpurificationequipmentAppendixConvertingfromlinearflow(cmhour)tovolumetricflowrates(mlmin)andviceversaAppendixConversiondata:proteins,columnpressuresAppendixTableofaminoacidsAppendixAnalyticalassaysduringpurificationAppendixStorageofbiologicalsamplesAdditionalreadingandreferencematerialOrderinginformationIntroductionBiomoleculesarepurifiedusingchromatographytechniquesthatseparatethemaccordingtodifferencesintheirspecificproperties,asshowninFigurePropertyTechniqueSizeGelfiltration(GF),alsocalledsizeexclusionChargeIonexchangechromatography(IEX)HydrophobicityHydrophobicinteractionchromatography(HIC)Reversedphasechromatography(RPC)Biorecognition(ligandspecificity)Affinitychromatography(AC)GelfiltrationHydrophobicinteractionIonexchangeAffinityReversedphaseFigSeparationprinciplesinchromatographypurificationFormorethanfortyyearssincetheintroductionofSephadex™,gelfiltrationhasplayedakeyroleinthepurificationofenzymes,polysaccharides,nucleicacids,proteinsandotherbiologicalmacromoleculesGelfiltrationisthesimplestandmildestofallthechromatographytechniquesandseparatesmoleculesonthebasisofdifferencesinsizeThetechniquecanbeappliedintwodistinctways:Groupseparations:thecomponentsofasampleareseparatedintotwomajorgroupsaccordingtosizerangeAgroupseparationcanbeusedtoremovehighorlowmolecularweightcontaminants(suchasphenolredfromculturefluids)ortodesaltandexchangebuffersHighresolutionfractionationofbiomolecules:thecomponentsofasampleareseparatedaccordingtodifferencesintheirmolecularsizeHighresolutionfractionationcanbeusedtoisolateoneormorecomponents,toseparatemonomersfromaggregates,todeterminemolecularweightortoperformamolecularweightdistributionanalysisGelfiltrationcanalsobeusedtofacilitatetherefoldingofdenaturedproteinsbycarefulcontrolofchangingbufferconditionsGelfiltrationisarobusttechniquethatiswellsuitedtohandlingbiomoleculesthataresensitivetochangesinpH,concentrationofmetalionsorcofactorsandharshenvironmentalconditionsSeparationscanbeperformedinthepresenceofessentialionsorcofactors,detergents,urea,guanidinehydrochloride,athighorlowionicstrength,at°CorinthecoldroomaccordingtotherequirementsoftheexperimentThishandbookdescribestheuseofgelfiltrationforthepurificationandseparationofbiomolecules,withafocusonpracticalinformationforobtainingthebestresultsThemediaavailable,selectioncriteriaandexampleswithdetailedinstructionsforthemostcommonapplicationsareincluded,aswellasthetheoreticalprinciplesbehindthetechniqueThefirststeptowardsasuccessfulseparationistoselectthecorrectmediumandthishandbookfocusesonthemostuptodategelfiltrationmediaandprepackedcolumnsThebiocompatibility,stabilityandutilityofgelfiltrationmediafromAmershamBioscienceshavemadetheseproductsthestandardchoiceinpracticallyeverylaboratoryusingthetechniqueAwidevarietyofprepackedcolumnsandreadytousemediaisavailableTheillustrationontheinsidecovershowstherangeofhandbooksfromAmershamBiosciencesthathavebeenproducedtoensurethatchromatographyandotherseparationtechniquesareusedeasilyandeffectivelyatanyscale,inanylaboratoryandforanyapplicationSymbolsthissymbolindicatesgeneraladvicewhichcanimproveproceduresorproviderecommendationsforactionunderspecificsituationsthissymboldenotesadvicewhichshouldberegardedasmandatoryandgivesawarningwhenspecialcareshouldbetakenthissymbolhighlightstroubleshootingadvicetohelpanalyseandresolvedifficultiesthatmayoccurchemicals,buffersandequipmentexperimentalprotocolCommonabbreviationsInchromatographyGF:gelfiltration(sometimesreferredtoasSEC:sizeexclusionchromatography)IEX:ionexchangechromatography(alsoseenasIEC)AC:affinitychromatographyRPC:reversephasechromatographyHIC:hydrophobicinteractionchromatographyCIPP:Capture,IntermediatePurificationandPolishingMPa:megapascalspsi:poundspersquareinchSDS:sodiumdodecylsulphateCIP:cleaninginplaceAnm,Anm:UVabsorbanceatspecifiedwavelengthMr:relativemolecularweight

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