His-tag-镍柱说明书

Instructions28-4020-89ADAffinitymediaGEHealthcareNiSepharose™6FastFlowCAutIon!Containsnickel.Mayproduceanallergicreaction.Immobilizedmetalionaffinitychromatography(IMAC)exploitstheinteractionbetweenchelatedtransitionmetalionsandside-chainsofcertainaminoacids(mainlyhistidine)onproteins.Ingeneral,Ni2+isthepreferredmetalionforpurificationofhistidine-taggedproteins.NiSepharose6FastFlowisaBioProcess™IMACmediumthatconsistsof90μmbeadsofhighlycross-linkedagarose,towhichachelatinggrouphasbeencoupled.Thischelatinggrouphasthenbeenchargedwithnickel(Ni2+)ions.NiSepharose6FastFlowhaslowNi2+leakage,highprotein-bindingcapacity,andiscompatiblewithawiderangeofadditivesusedinproteinpurification.Itshighflowpropertiesmakeitexcellentforscale-up.NiSepharose6FastFlowisavailablein5,25,100,500ml,1L,and5Lbulkpacks.NiSepharose6FastFlowisalsoavailableprepackedin1and5-mlHisTrap™FF,1and5-mlHisTrapFFcrudeand20-mlHisPrep™FF16/10columns.p.2Contents1.Productdescription32.Generalconsiderations53.Packingcolumns64.Packinglab-scalecolumns75.Packingprocess-scalecolumns86.Evaluationofcolumnpacking87.Preparationbeforepurification118.Purificationprocedure139.Optimization1410.Troubleshooting1511.Regeneratingthemedium1812.Cleaning-in-Place(CIP)1913.Storage2014.Furtherinformation2015.Orderinginformation20p.31.ProductdescriptionNiSepharose6FastFlowishighlystableandcompatiblewithawiderangeofcommonadditives.Thishelpstomaintainbiologicalactivityandincreaseproductyield,whileatthesametimegreatlyexpandingtherangeofsuitableoperatingconditions.Inaddition,themediumiseasytopackanduse,anditshighflowpropertiesmakeitexcellentforscaling-up.ThekeycharacteristicsofthemediumarelistedinTable1.AvarietyofcompoundsthatarecompatiblewithNiSepharose6FastFlowarelistedinTable2.table1.MediumcharacteristicsMatrixHighlycross-linked6%sphericalagaroseDynamicbindingcapacity*Approx.40mghistidine-taggedprotein/mlmediumMetalioncapacityApprox.15μmolNi2+/mlmediumAverageparticlesize90μmMax.linearflowrate†600cm/h(20ml/min)usingXK16/20columnwith5cmbedheightRecommendedflowrate†150cm/hMax.operatingpressure†0.1MPa,1bar(whenpackedinXKcolumns.columns.Mayvaryifusedinothercolumns)Chemicalstability‡Stablein:0.01MHCl,0.1MNaOH.Testedfor1weekat40°C.1MNaOH,70%aceticacid.Testedfor12hours.2%SDS.Testedfor1hour.30%2-propanol.Testedfor30min.pHstability‡Shortterm(atleast2hours)2–14Longterm(≤1week)3–12Storage20%ethanolStoragetemperature4°Cto30°C*Dynamicbindingcapacityconditions:Sample:1mg/mlhistidine-taggedpureproteins(Mr43000)inbindingbuffer(capacityat10%breakthrough)orhistidine-taggedprotein(Mr28000)boundfromE.coliextract.Columnvolume:0.25mlor1mlFlowrate:0.25ml/minor1ml/min,respectivelyp.4Bindingbuffer:20mMsodiumphosphate,0.5MNaCl,5mMimidazole,pH7.4Elutionbuffer:20mMsodiumphosphate,0.5MNaCl,0.5Mimidazole,pH7.4note:Dynamicbindingcapacityisprotein-dependent.†H2Oatroomtemperature.‡Ni2+-strippedmedium.table2NiSepharose6FastFlowiscompatiblewiththefollowingcompounds,atleastattheconcentrationsgivenReducingagents*5mMDTE5mMDTT20mMß-mercaptoethanol5mMTCEP10mMreducedglutathioneDenaturingagents8Murea†6MGua-HCl†Detergents2%Triton™X-100(nonionic)2%Tween™20(nonionic)2%NP-40(nonionic)2%cholate(anionic)1%CHAPS(zwitterionic)otheradditives500mMimidazole20%ethanol50%glycerol100mMNa2SO41.5MNaCl1mMEDTA‡60mMcitrate‡Buffersubstances50mMsodiumphosphate,pH7.4100mMTris-HCl,pH7.4100mMTris-acetate,pH7.4100mMHEPES,pH7.4100mMMOPS,pH7.4100mMsodiumacetate,pH4†*SeeGeneralconsiderations.†Testedfor1weekat40°C.‡ThestrongchelatorEDTAhasbeenusedsuccesfullyinsomecasesat1mM.Generally,chelatingagentsshouldbeusedwithcaution(andonlyinthesample,notinbuffers).Anymetal-ionstrippingmaybecounteractedbyadditionofasmallexcessofMgCl2beforecentrifugation/filtrationofthesample.Notethatstrippingeffectsmayvarywithappliedsamplevolume.p.52.GeneralconsiderationsNiSepharose6FastFlowissuppliedprechargedwithNi2+ions.Ingeneral,Ni2+isthepreferredmetalionforpurificationofrecombinanthistidine-taggedproteinsandimidazoleisusedforelution.ElutionusingreducedpH(linearorstepwisedecreaseinpH)isaalternativeelutionprocedure.Imidazoleatlowconcentrationsiscommonlyusedinthebindingandwashbuffertominimizebindingofunwantedhostcellproteins.Forthesamereason,itisimportanttoalsoincludeimidazoleinthesample(generally,atthesameconcentrationasinthewashbuffer).Atsomewhathigherconcentrations,imidazolemayalsodecreasethebindingofhistidine-taggedproteins.Theimidazoleconcentrationmustthereforebeoptimizedtoensurethebestbalanceofhighpurity(lowbindingofunwantedproteins)andhighyield(bindingofallofthehistidine-taggedprotein).Theconcentrationofimidazolethatwillgiveoptimalpurificationresultsisprotein-dependent,andisusuallyslightlyhigherforNiSepharose6FastFlowthanforsimilarIMACmediaonthemarket.(seeDataFile11-0008-86andOptimization).LeakageofNi2+fromNiSepharose6FastFlowislowunderallnormalconditions(lowerthanforotherIMACmediatested).Forapplicationswhereverylowleakageduringpurificationiscritical,leakagecanbediminishedevenfurtherbyperformingablankrunoralternativelyperforminganacidicwash(seePurificationprocedure,pa