Ni-sepharose-6-Fast-Flow说明书

Instructions11-0008-87AEAffinitymediaGEHealthcareNiSepharose™6FastFlowImmobilizedmetalionaffinitychromatography(IMAC)exploitstheinteractionbetweenchelatedtransitionmetalionsandside-chainsofcertainaminoacids(mainlyhistidine)onproteins.Ingeneral,Ni2+isthepreferredmetalionforpurificationofhistidine-taggedproteins.TheIMACmediumNiSepharose6FastFlowconsistsof90µmbeadsofhighlycross-linkedagarose,towhichachelatinggrouphasbeencoupled.Thischelatinggrouphasthenbeenchargedwithnickel(Ni2+)ions.NiSepharose6FastFlowhaslowNi2+leakage,highprotein-bindingcapacity,andiscompatiblewithawiderangeofadditivesusedinproteinpurification.Itshighflowpropertiesmakeitexcellentforscale-up.Themediumisalsosuitableforgravityflowcolumnpurificationandmultiwellplatescreening.NiSepharose6FastFlowisavailablein5,25,100and500mllabpacks,prepackedin1-mlHisGraviTrap™gravity-flowcolumnsandHisTrap™FF1and5-ml,HisTrapFFcrude1and5-mland20-mlHisPrep™FF16/10columnsforuseinchromatographysystemsuchasÄKTA™designorFPLC™System.Caution!Containsnickel.Mayproduceanallergicreaction.Pleasereadtheseinstructionscarefullybeforeuse.Payextraattentiontothechapters“Generalconsiderations”and“Preparationbeforepurification”.p.2ProductdescriptionNiSepharose6FastFlowishighlystableandcompatiblewithawiderangeofcommonadditives.Thishelpstomaintainbiologicalactivityandincreaseproductyield,whileatthesametimegreatlyexpandingtherangeofsuitableoperatingconditions.Inaddition,themediumiseasytopackanduse,anditshighflowpropertiesmakeitexcellentforscaling-up.ThekeycharacteristicsofthemediumarelistedinTable1.AvarietyofcompoundsthatarecompatiblewithNiSepharose6FastFlowarelistedinTable2.TableofcontentsProductdescription2Generalconsiderations5Columnpacking6Preparationbeforepurification8Purificationprocedureforapackedcolumn11Batch/gravity-flowpurification12Optimization14Troubleshooting15Regeneratingthemedium18Cleaning-in-Place(CIP)19Storage20Furtherinformation20Orderinginformation21p.3Table1.MediumcharacteristicsMatrixHighlycross-linkedsphericalagarose,6%Dynamicbindingcapacity*Approx.40mg(histidine)6-taggedprotein/mlmediumMetalioncapacityApprox.15µmolNi2+/mlmediumAverageparticlesize90µmMax.linearflowrate†600cm/h(20ml/min)usingXK16/20columnwith5cmbedheightRecommendedflowrate†150cm/hMax.operatingpressure†0.1MPa,1bar(whenpackedinXKcolumns.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/ml(histidine)6-taggedpureproteins(Mr43000)inbindingbuffer(capacityat10%breakthrough)or(histidine)6-taggedprotein(Mr28000)boundfromE.coliextract.Columnvolume:0.25mlor1mlFlowrate:0.25ml/minor1ml/min,respectivelyBindingbuffer:20mMsodiumphosphate,0.5MNaCl,5mMimidazole,pH7.4Elutionbuffer:20mMsodiumphosphate,0.5MNaCl,500mMimidazole,pH7.4Note:Dynamicbindingcapacityisprotein-dependent.†H2Oatroomtemperature.‡Ni2+-strippedmedium.p.4Table2.NiSepharose6FastFlowiscompatiblewiththefollowingcompounds,attheconcentrationsgivenReducingagents*5mMDTE5mMDTT20mMß-mercaptoethanol5mMTCEP10mMreducedglutathioneDenaturingagents8Murea†6MGua-HCl†Detergents2%Triton™X-100(nonionic)2%Tween™20(nonionic)2%NP-40(nonionic)2%cholate(anionic)1%CHAPS(zwitterionic)Otheradditives20%ethanol50%glycerol100mMNa2SO41.5MNaCl1mMEDTA‡60mMcitrate‡Buffersubstances50mMsodiumphosphate,pH7.4100mMTris-HCl,pH7.4100mMTris-acetate,pH7.4100mMHEPES,pH7.4100mMMOPS,pH7.4100mMsodiumacetate,pH4†*SeeGeneralconsiderationsp.5andBlankrun,p.11.†Testedfor1weekat40°C.‡ThestrongchelatorEDTAhasbeenusedsuccessfullyinsomecasesat1mM.Generally,chelatingagentsshouldbeusedwithcaution(andonlyinthesample,notinbuffers).Anymetal-ionstrippingmaybecounteractedbyadditionofasmallexcessofMgCl2beforecentrifugation/filtrationofthesample.Notethatstrippingeffectsmayvarywithappliedsamplevolume.p.5GeneralconsiderationsNiSepharose6FastFlowissuppliedprechargedwithNi2+ions.Ingeneral,Ni2+isthepreferredmetalionforpurificationofrecombinanthistidine-taggedproteinsandimidazoleisusedforelution.Imidazoleatlowconcentrationsiscommonlyusedinthebindingandwashbuffertominimizebindingofunwantedhostcellproteins.Forthesamereason,itisimportanttoalsoincludeimidazoleinthesample(generally,atthesameconcentrationasinthewashbuffer).Atsomewhathigherconcentrations,imidazolemayalsodecreasethebindingofhistidine-taggedproteins.Theimidazoleconcentrationmustthereforebeoptimizedtoensurethebestbalanceofhighpurity(lowbindingofunwantedproteins)andhighyield(bindingofallofthehistidine-taggedprotein).Theconcentrationofimidazolethatwillgiveoptimalpurificationresultsisprotein-dependent,andisusuallyslightlyhigherforNiSepharose6FastFlowthanforsimilarIMACmediaonthemarket.(seeDataFile11-0008-86andOptimization).LeakageofNi2+fromNiSepharose6FastFlowislowunderallnormalconditions(lowerthanforotherIMACmediatested).Forapplicationswhereverylowleakageduringpurificationiscritical,leakage