TALON Metal Affinity Resins User Manual - … · 2017-09-12 · Using theTALON® Metal Affinity...

TALON®

Metal Affinity ResinsUser Manual

PT1320-1 (PR6Z2142)Published 25 April 2007

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT1320-1 2 Version No. PR6Z2142

TALON® Metal Affinity Resins User Manual

Table of Contents

I. Introduction 4

II. List of Components 13

III. Additional Materials Required 15

IV. Buffers for TALON® Purification & Buffer Kits 20

V. Buffers for TALON® Magnetic Beads 21

VI. Transformation & Protein Expression 22 A. TransformationofHostCellswithExpressionVectors 22 B. ProteinExpression 22

VII. Sample Preparation 23

A. TALON®xTractorBufferSamplePreparation 23

B. StandardSamplePreparationtoIsolateNativeProteins 23

C. StandardSamplePreparationtoIsolateDenaturedProteins 24

D. StandardSamplePreparationforTALON®CellThruResin 25

E. StandardHT96-WellSamplePreparation 26

F. StandardSamplePreparationforTALON®MagneticBeads 26

G. SamplePreparationDirectlyfromOvernightCulturesfor

TALON®MagneticBeads 27

VIII. Protein Purification Protocols 28

A. GeneralInformation 28

B. Batch/Gravity-FlowColumnPurification 30 TALON®Resin,SuperflowResin,orCellThruResin

C. Large-ScaleBatchPurification 32 TALON®Resin,SuperflowResin,orCellThruResin

D. Medium-PressureColumnPurification 33 TALON®SuperflowResin

E. 5mlTALON®SingleStepColumnPurification 34

F. 20mlTALON®SingleStepColumnPurification 36

G. TALONspin™ColumnPurification 38

H. TALON®HT96-WellPurificationProtocol 40

I. TALON®MagneticBeadsPurificationProtocol 43

IX. Resin Washing, Reuse, and Storage 46

X. Troubleshooting Guide 48

XI. References 53

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Table of Contents continued

Notice to Purchaser

Clontechproductsaretobeusedforresearchpurposesonly.Theymaynottobeusedforanyotherpurpose,including,butnotlimitedto,useindrugs,in vitrodiagnosticpurposes,therapeutics,orinhumans.Clontechproductsmaynotbetransferredtothirdparties,resold,modifiedforresale,orusedtomanufacturecommercialproductsortoprovideaservicetothirdpartieswithoutwrittenapprovalofClontechLaboratories,Inc.

LicensedunderU.S.PatentNo.4,569,794anditsinternationalequivalentsforuseinresearchrelatedbiopolymers.Licenses forcommercialapplicationsareavailable fromIndianaPro-teomicsConsortium,Inc.(Inproteo).

TALON®ResinproductsarecoveredunderU.S.PatentNo.5,962,641.

Sepharose®isaregisteredtrademarkofGEHealthcare.

Triton™isatrademarkofTheDowChemicalCompany.

Superflow™,Uniflow™,andCellThru™aretrademarksofSterogeneBioseparations,Inc.Clontech,ClontechlogoandallothertrademarksarethepropertyofClontechLaboratories,Inc.,unlessnotedotherwise.ClontechisaTakaraBioCompany.©2007

Appendix A. ReagentCompatibilitiesandIncompatibilities 54

Appendix B.Mini-ScaleProteinPurificationProtocolforTALON®or TALON®SuperflowResin 56

Appendix C.VectorInformation 58List of Figures

Figure1. SchematicdiagramoftheTALON®IMACSystem. 5

Figure2. Elutionmechanismofrecombinantpolyhistidine-taggedproteinsfromTALON®Resin. 6

Figure3. BindingofhistidinestotheTALON®Resinmetalion. 6

Figure4. UsingtheTALON®MetalAffinityResinsUserManual 7

Figure5. Purificationofpolyhistidine-taggedproteinsusingTALON®Resin 19

Figure6. pHAT10/11/12combinedvectormapandMCS. 58

Figure7. pHAT20combinedvectormapandMCS. 59

List of TablesTableI. ProteinpurificationusingTALON®Resins. 10

TableII. TALON®Resincharacteristics 12

TableIII. Reagentcompatibility 54



I. Introduction

Proteinshaveevolvedverycomplexstructuresinordertoperformadiversear-rayoffunctions.Asaresult,theirphysicochemicalpropertiesvarygreatly,pos-ingdifficultiesfordevelopingversatilepurificationprotocols.Onewaytocir-cumventthisproblemistoincorporateapurificationtagintotheprimaryaminoacidsequenceofatargetprotein,thusconstructingarecombinantproteinwithabindingsitethatallowspurificationunderwell-defined,genericconditions.

Immobilized Metal Affinity Chromatography (IMAC)IMACwasintroducedin1975asagroup-specificaffinitytechniqueforsepa-ratingproteins(Porathet al.,1975).Theprincipleisbasedonthereversibleinteractionbetweenvariousaminoacidsidechainsandimmobilizedmetalions.Dependingontheimmobilizedmetal ion,differentsidechainscanbe involved in theadsorptionprocess.Mostnotably,histidine,cysteine,andtryptophansidechainshavebeenimplicatedinproteinbindingtoim-mobilizedtransitionmetalionsandzinc(Figure1,Porath,1985;Sulkowski,1985;Hemdan&Porath,1985a;Hemdan&Porath,1985b;Zhaoet al.,1991).

TALON® IMAC ResinsTALON®Resinsaredurable,cobalt-basedIMACresinsdesignedtopurifyrecombinantpolyhistidine-taggedproteins(Bushet al.,1991).Theseresinsarecompatiblewithmanycommonlyusedreagents(AppendixA),andallowpro-teinpurificationundernativeordenaturingconditions.Theycanbeusedwithallprokaryoticandeukaryoticexpressionsystemsinavarietyofformats,in-cludingsmall-(ormini-)scalebatchscreening,large-scalebatchpreparations,andmethodsusinggravity-flowcolumnsandspincolumns.Inaddition,pro-tocolsusedwithNi+2-basedIMACcolumnsusuallyworkwithTALON®resins.

TALONMagneticBeadsareagarosebeadsutilizingourpatentedTALONtech-nology.ThebeadscombinetheadvantageofhighlyselectiveTALONchemistrywithmagneticbeadseparation.Magneticparticlesinthebeadsfacilitatequickandeasypurificationofproteinsatmicroscalelevelusingamagneticseparator.MicroscalepurificationwithTALONMagneticBeadscanbeusedforscreeningofexpressionlevelsorforprotein-proteininteractionstudies.

Tetradentate metal chelatorToovercometheproblemofmetalleakageencounteredwithotherIMACres-ins,TALON®Resinutilizesaspecialtetradentatemetalchelatorforpurifyingrecombinantpolyhistidine-taggedproteins(U.S.PatentNo.5,962,641).Thischelatortightlyholdstheelectropositivemetalinanelectronegativepocket(Figure1),whichisidealforbindingmetalionssuchascobalt.ThebindingpocketisanoctahedralstructureinwhichfourofthesixmetalcoordinationsitesareoccupiedbytheTALONResinligand.ThisprocessenhancestheproteinbindingcapacityofTALONResinbymakingtheboundmetalionac-cessibletosurroundingpolyhistidine-taggedproteins.Thetetradentatemetalbindingmeansthatnometallossoccursduringproteinpurificationunderrec-

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ommendedconditions,eveninthepresenceofstrongdenaturantssuchas6Mguanidinium.SuchdurabilityallowsTALONResintobereused(SeeSectionIX).

Cobalt IMAC Resin permits milder elution conditionsTALONResinexhibitssubtleyetimportantdifferencesinbindingofpolyhis-tidine-taggedproteinswhencomparedwithnickelIMACresins.Forexample,nickel-basedIMACresinsoftenexhibitanundesirabletendencytobindun-wantedhostproteinscontainingexposedhistidineresidues(Kasheret al.,1993).WhileTALONResinbindspolyhistidine-taggedproteinswithenhancedselectivityovernickel-basedresins,itexhibitsasignificantlyreducedaffin-ityforhostproteins.Thisbehaviorofferstwopracticaladvantages.First,virtuallynobackgroundproteinsareboundtotheresinwhenthesampleisapplied;consequently,cumbersomewashingproceduresarenotgenerallyrequiredbeforeproteinelution.Second,polyhistidine-taggedproteinselutefromtheresinunderslightlylessstringentconditions—aslightlyhigherpHor lower imidazoleconcentration—thanwithnickel IMACresins.Elutionoccurswhentheimidazolenitrogen(pKaof5.97)isprotonated(Figure2),generatingapositivelychargedammoniumion,whichisrepelledbythepositivelychargedmetalatom.Alternatively,theboundpolyhistidine-taggedproteincanbecompetitivelyelutedbysimplyaddingimidazoletotheelu-tionbuffer,becauseimidazoleisidenticaltothehistidinesidechain.

Figure 1. Schematic diagram of the TALON® IMAC System.Part A.TALONMetalAffinityResin;ASepharosebeadbearingthetetradentatechelatoroftheCo2+metalion.Part B.Thepolyhi-stidine-taggedrecombinantproteinbindstotheresin.

I. Introduction continued

HC

N

N

N

N

N

H2C

H2C

COO–

COO–

COO–

Co2+SepharoseBead

A B



Polyhistidine affinity tagsHistidinesexhibithighlyselectivecoordinationwithcertaintransitionmet-alsandhavegreatutilityinIMAC.UnderconditionsofphysiologicalpH,histidinebindsbysharingelectrondensityoftheimidazolenitrogenwiththe electron-deficient orbitals of transition metals (Figure 3). Althoughthree histidines may bind transition metals under certain conditions,six histidines reliably bind transition metals in the presence of strongdenaturants such as guanidinium (Hochuli et al., 1987). Such proteintags are commonly referred to as“6 x histidine,”“hexaHis,” or“(His)6.”

HAT—a novel IMAC affinity tag

With the advent of recombinant genetic technologies, the design andproduction of recombinant proteins containing novel polyhistidine tagsontheirN-orC-terminihasbecomemorestraightforward(Hochuliet al.,1987;Hochuliet al.,1988).TheHATsequence(patentpending)isanovelIMACaffinitytagderivedfromauniquenaturalproteinsequence(Chagaet al.,1999).Itcontainssixhistidinesunevenlyinterleavedbyotheraminoacidresidues(seeAppendixC).TheHATaminoacidsequenceisderivedfromtheN-terminusofchickenmusclelactatedehydrogenase—asequencethatisuniqueamongreportedproteinsequences.Thenoveltagdoesnothave theexcessivepositivechargecharacteristicof thecommonlyused6xhistidinetag,thuscontributingtobettersolubilityofHAT-fusionpro-teinsandsimilaraffinity towards immobilized transitionmetal ionsandzinc.ClontechofferstheHATProteinExpressionandPurificationSystem(Cat. No. 631205)—a complete system containing reagents and vectors

Figure 2. Elution mechanism of recombinant polyhistidine-tagged proteins from TALON® Resin. Elutionoccurswhentheimidazoleni-trogen(pKa=5.97)isprotonated,generatingapositivelychargedammoniumionwhichisrepelledbythepositivelychargedmetalion.Alternatively,theboundpolyhistidine-taggedproteincanbecompetitivelyelutedbyaddingimidazoletotheelutionbuffer.

Figure 3. Binding of histidines to the TALON® Resin metal ion.UnderconditionsofphysiologicalpH,histidinebindsbysharingimidazolenitrogenelectrondensitywiththeelectron-deficientorbitalsofthemetalion.


N

N

N

N

2 +

H +

N

N H

? Unprotonated Histidine

binds to metal

N

N H

H

Protonated Histidinerepelled by metal

+



Protein Purification Strategy (Section I) • Protein Purification Methods - Resin Characteristics • Choosing Buffers • Elution Strategy

Buffers (Sections III & IV) Native Denaturing

Protein Expression (Section VI) A. Transformation B. Protein Expression

Sample Preparation (Section VII)

Native Purification Denaturing PurificationA. xTractor Buffer C. Standard &B. Standard & Superflow Resin Superflow Resin D. CellThru ResinD. CellThru Resin F. TALON Magnetic E. High-throughput (96-well) BeadsF. & G. TALON Magnetic Beads

Protein Purification (Section VIII) B. Batch or Gravity FlowC. Large-Scale Batch

D. Medium-Pressure & FPLC Column E. 5 ml Single Step ColumnsF. 20 ml Single Step Columns

G. TALONspin™ Columns H. HT 96-Well Plate I. TALON Magnetic Beads Appendix B. Mini-Scale

Figure 4. Using the TALON® Metal Affinity Resins User Manual.Overviewoftheprocedures.


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designedforbacterialexpressionandpurificationofHAT(histidineaffinitytag)proteins.Eachofthethreevectors—pHAT10,pHAT11,andpHAT12—containamultiplecloningsite(MCS)inallthreeframestoallowcloningoftargetcDNA.(ForvectormapandMCS,seeAppendixCofthisUserManual.)AconvenientlylocatedenterokinaseproteolyticsitebetweentheHATsequenceandtheMCSprovidesameansforremovingtheaffinitytag.

Formoreinformation,seetheHATExpression&PurificationSystemUserManual(PT3250-1),whichcanbedownloadedfromourwebsiteatwww.clontech.com.

TALON® Express Bacterial Expression and Purification Kits

TALONExpressBacterialExpressionandPurificationKits aredesignedforthecloning,expression,andpurificationofpolyhistidine-taggedproteinsusing E. coli.ThekitscontaintwoseparatebacterialexpressionvectorsencodingN-orC-terminal6xHNfusiontags.TheseIPTG-inducible,pET-basedvectorsprovidehighlevelsofproteinexpression.TheexpressedproteinsarereadyforquickandeasypurificationusingtheTALONresinandbuffersprovidedinthekits.

Overview of TALON® Resins

Thefollowingisalistofdifferentresinformatstomeetyourpurificationneeds.

• TALON® Metal Affinity Resin is useful for batch and low-pressurechromatographicapplications.This resinutilizesSepharoseCL-6B (GEHealthcare),adurablesubstrate thatperformsverywellundernativeanddenaturingconditionsincentrifuge-mediatedpurificationschemes.Thelargeporesizeresinhasahigh-bindingcapacity.Thisresinisalsoavailablepre-packedin2mlgravitycolumns.

• TALON® Superflow Resinisusefulforarangeofapplications,includingmediumpressureapplicationswithFPLCsystemsatbackpressuresofupto150psi(1MPa)andhighflowratesupto5mlpercm2permin.Thisresinisrecommendedifshortpurificationtimesareessential,orifpurificationprotocolsdevelopedatbenchscalewillbescaledupforlargervolumes.

ThisresinutilizesSuperflow-6(SterogeneBioseparations,Inc.),anaga-rose-basedmediumfeaturingauniquepolysaccharidecompositionthatresistsbiologicaldegradation.Superflow-6beadsarealsostabilizedbyachemicalcross-linkingreactionthatallowsflowratesupto10timeshigherthanarepossiblewithregularcross-linkedbeads.

TheTalon®SuperflowResinisalsopresentinthehighthroughput(HT)96-wellplate.




• TALON® CellThruResinisanovelIMACresinforpurifyingpolyhistidine-taggedproteinsfromcrudecelllysates,sonicates,andfermentationliquids.ThelargerbeadsizeofTALONCellThruResin(300–500µm)permitscellulardebristoflowthroughthecolumn,eliminatingtheneedforhigh-speedcentrifugation.DestabilizingfactorsareremovedmorequicklywiththisresinthanwithotherIMACresins,becausethenumberofstepsarereduced.

CellThru2ml&10mlDisposableColumnshavealargefilterporesize(90–130µm)thatallowscellulardebristoflowthroughthecolumnduringthepurificationprocess.The2mlcolumnsaresuitablefor1–2mlbedvolumes,whilethe10mlcolumnsaresuitablefor5–10mlbedvolumes.

• TALONspin™ Columns areidealforrapidlyandsimultaneouslypurifyingsmallamountsofpolyhistidine-taggedproteins.Thesecolumnsarerecom-mendedforsingle-useapplicationsorforuseasminigravity-flowcolumns.Eachcolumncontains0.5mlofTALON-NXResin,whichisoptimizedforperformanceinaspincolumn.Eachcolumnwillyield2–4mgofpolyhi-stidine-taggedprotein;exactyieldswillvarywithconditionsusedandpolyhistidine-taggedproteincharacteristics.Inaddition,yieldandpuritywilldependuponexpressionlevelandlysateconcentration.Beginningwiththeclarifiedsample,theentireproceduretakesapproximately30min.

• TALON® Magnetic Beads areusefulformicroscalepurificationofpolyhisti-dine-taggedproteinsundernativeordenaturingconditions.Thebeadscanalsobeusedtopurifyproteinsdirectlyfromcleared(centrifuged)orcrudecelllysates.Forscreeningofexpressionlevels,proteinscanbepurifieddirectlyfromovernightculturesassmallas0.5ml(dependingontheexpres-sionlevel).TheuseofTALONchemistryallowsforseamlessscaling-uptolarge-scalepurificationoftargetproteinsusingourstandardTALONresin.

TALONMagneticBeadsaresuppliedasa5%suspensionin25%ethanol,availableineithera2x1mlor6x1mlformat.Thebeadshaveabindingcapacityof750µgof6xHN-taggedAcGFPper1mlofsuspension.Whenperformingassaysinsingletubes,100–200µlofbeadsaresufficientforeachassay.Smalleramountsofbeadsmaybeused,buttheremaybedifficultiesinhandlingthebeadsinsmallbuffervolumes.




TABLE I. PROTEIN PURIFICATION USING TALON® RESINS

Method Application Key Benefit

TALON®Metal Affinity Resin or TALON® Superflow ResinMini-Scale •Checkforpresenceoftaggedprotein•Fast(AppendixB) •Estimateexpressionlevels •Requiresonly1mlofcell •Testbufferconditions culture+1mlofresin

Batch/Gravity •Purify>5mgoftaggedprotein •VeryhighpurityFlow Column using1mlofresin •Doesnotrequire(Sec.VIII.B) pressurizedcolumn equipment

Large-Scale •Large-andproduction-scale •Fasterthanprotocolsthat(Sec.VIII.C&D) purification;easytoscaleup usegravity-flowcolumns •Higherpuritythanusing batchprocessaloneTALON® CellThru ResinBatch/Gravity •Forpurifyingproteinsfrom •FastFlow Column & nonclarifiedcelllysates,sonicates, •Doesnotrequirehigh-Large-Scale orfermentationliquids speedcentrifugation(Sec.VIII.B&C) TALON® Single Step Columns (5 ml, 20 ml) Miniprep •Processseveraldifferentsamples •Fast(~30–40min)1

(Sec.VIII.E&F) simultaneously •Usesunlysedcellculture •Lysebacterialcellsandbind •Simplifiesscreeningof histidine-taggedproteininonestep multipleproteins •Obtain0.2–0.6mg(5mlcolumn) •Ready-to-usecolumns or0.5–4mg(20mlcolumn).TALONspin™ Columns Spin Column •Processseveraldifferentsamples •Fast(~30min)2

(Sec.VIII.G) simultaneously •Usesonly0.6–1mlof •Obtain2–4mgofpurifiedprotein cellculturelysate perspincolumn •Ready-to-usecolumnsTALON® HT 96-Well Plates96-Well Plates •High-throughputprocessingofsamples•Fast(<30min)2

(Sec.VIII.H) •Obtainupto1.0mgofpurifiedprotein•Usesupto2mlof perwell crudelysateperloadTALON®Magnetic BeadsMagnetic Beads•Microscalepurification •Fast(Sec.VII.F&G •Checkforpresenceoftaggedprotein •Requires0.5mlcultureandSec.VIII.I) •Estimateexpressionlevels •Doesnotrequirehigh- •Purifyfromcrudeuncleared speedcentrifugation celllysatesorcultures •Amenabletohigh-throughput1Includestimeforsampleprepandpurification.2Startingwithclarifiedlysate;doesnotincludetimetopreparesamples.




Protein Purification Methods Using TALON® ResinThefollowinggeneralguidelinesareusedforpurifyingpolyhistidine-taggedproteinfromtransformedE. colicultures.Figure4andTableIprovideanoverviewofTALONResinproteinpurificationmethodsandapplications.Chooseamethodthatbestsuitsyourresearchneeds.

• Use2mlof resinsuspensionper~3mgofanticipatedpolyhistidine-taggedprotein.2mlofhomogeneouslyresuspendedresinwillprovide1ml(bedvolume)ofTALONResin.

• Thebuffersandpurificationconditionsshouldworkwellformostsoluble,monomericproteinsexpressedinE. coli.

• Initially,testeachdifferentexpressionsystemandpolyhistidine-taggedproteininsmall-scalebatchpurificationtodetermineexpressionlevelsandtooptimizetheprotocol.TALONSingleStepColumnsaredesignedforthistypeofanalysis(SectionVIII.E&F).Alternatively,Amini-scalebatchpurificationprotocolisprovidedinAppendixB;oryoucanuseaTALONspinColumn(SectionVIII.G).

• Purificationmethodsthatworkwithnickelorzinc-basedIMACresinsshouldalsoworkwiththeseresins.However,someoptimizationmayberequired.

Note:TALONresinhasbeenoptimizedandshouldonlybeusedwiththebufferformula-tionsoutlinedinthisusermanualforoptimalperformance.

Choosing the Buffers: Imidazole Versus pH Gradient

TALONResinpurificationschemestypicallyuseeitheranimidazoleorapHgradientforwashingandelution.ImidazoleintheEquilibrationand/orEquilibration/WashBuffersminimizesnonspecificbindingandreducestheamountofcontaminatingproteins.Thus,werecommendfirstpurifyingpoly-histidine-taggedproteinsusinganimidazolegradient.However,imidazoleandpolyhistidine-taggedproteinsabsorbat280nmandelutionpeaksmaybedifficulttodetectspectrophotometrically,especiallyifyouarepurifyingsmallamountsofpolyhistidine-taggedproteins.Inthesecases,collecttheleadingedgeoftheimidazolebreakthroughpeakandcheckforpolyhistidine-taggedproteinsbyaproteinspecificassay(Bradford,1976)andSDS/PAGE.Alternatively,useapHgradienttopurifypolyhistidine-taggedproteinsthatarestablefrompHrange5.0–7.0.SeeSectionIIIforbuffercompositions.

Elution Strategy: Step Versus Linear Gradients Inmostcases,stepgradientsarepreferredoverlineargradients,becauselineargradientsleadtobroadelutionpeaks,whichcandilutetheproductandmakedetectiondifficult.Scaling-upstepgradientsisalsolesscompli-catedthanscaling-uplineargradients.




TABLEII.TALON®RESINCHARACTERISTICS

TALON® TALON® TALON® TALONspin™Feature Resin Superflow CellThru Column

Capacity1 5–15 5–20 5–10 2–4(mgprotein/mlresin)

Matrix SepharoseCL-6B Superflow Uniflow Sepharose6B

Beadsize(µm) 45–165 60–160 300–500 16–24

Max.Linear 75–150 3,000 800 n/a2flowrate(cm/hr)

Max.Volume 0.5–1.0 50 13 n/aflowrate3(ml/min)

Max.Pressure 2.8psi 140psi 9psi n/a 0.2bar 10bar 0.62bar n/a 0.02MPa 0.97MPa 0.06MPa n/a

pHstability 2–14(2hr) 2–14(2hr) 2–14(2hr) 2–8.5(2hr) 3–14(24hr) 3–14(24hr) 3–14(24hr) 2–7.5(24hr)

Proteinexclusion 4x107 4x106 2x107 n/alimit(Da)1Thebindingcapacityforindividualproteinsmayvary.EachoftheabovementionedTALONproductshas

differentapplications.PleaserefertoTableIforapplicationsandbenefits.2n/a=notapplicable3Determinedona5x1cmcolumn.




II. List of Components

TALONResin,TALONSuperflowResin,andTALONCellThruResinaresuppliedas50%(w/v)slurriesinnonbuffered20%ethanol.Pleasenotethatduringshippingandstorage,theresinwillsettle;thus,werecommendthatyouthoroughlyresuspenditbeforealiquotting.2mlofhomogeneouslyresuspendedresinwillprovide1mlofTALONResinwithabindingcapacityofatleast5mgofpolyhistidine-taggedprotein.

Store all of these resins, columns and buffers at 4°C unless otherwiseindicated.Do not freeze TALON® Resins.

• TALON® Metal Affinity Resin Cat. No. Amount 635501 10ml 635502 25ml 635503 100ml 635504 250ml

• TALON® Superflow Resin Cat. No. Amount 635506 25ml 635507 100ml

• TALON®Single Step Columns (5ml,Cat.Nos.635628&635631; &20ml,Cat.No.635632) ThesecolumnscontainadrymixtureofTALONCellThruresin andxTractor

Buffertoextractandbindhistidine-taggedproteinsinonestep.

• TALONspin™Columns (Cat.Nos.635601,635602,635603) Thesecolumnscontain0.5mlofTALON-NXresin asa50%suspension

innonbuffered20%ethanol.

• TALON® HT 96-Well Plate (Cat.No.635622) 1 TALON96-WellPlate 1 PlateTopSeal 1 PlateBaseSeal 1 CollectionPlate

• TALON® Magnetic Beads(Cat.Nos.635636&635637) Cat. No. Amount 635636 2x1ml 635637 6x1ml

• TALON® Magnetic Beads Buffer Kit (Cat.No.635638) 60ml 5XEquilibration/WashBuffer 15ml 4XElutionBuffer 30ml 1XxTractorBuffer



II. List of Components continued

• TALON® CellThru Resin Cat. No. Amount 635509 10ml 635510 100ml

• TALON® CellThru Disposable Columns Cat. No. Size 635512 50x2mlcolumn 635513 20x10mlcolumn

• TALON® 2 ml Disposable Gravity Columns (Cat.No.635606)

• TALON® Purification Kit(Cat.No.635515)

10ml TALON®MetalAffinityResin 160ml 5XEquilibration/WashBuffer (250mMsodiumphosphate,1.5MNaCl,pH7) 160ml 5XEquilibrationBuffer (250mMsodiumphosphate,1.5MNaCl,pH8) 25ml 10XElutionBuffer (1.5Mimidazole,pH7) 5 2mlDisposableGravityColumns 1 10mlDisposableGravityColumn

• TALON® Buffer Kit (Cat.No.635514)

160ml 5XEquilibration/WashBuffer (250mMsodiumphosphate,1.5MNaCl,pH7) 160ml 5XEquilibrationBuffer (250mMsodiumphosphate,1.5MNaCl,pH8) 25ml 10XElutionBuffer (1.5Mimidazole,pH7)

• TALON® xTractor Buffer Kit (Cat.No.635623)

StoreDNaseIat–20°C.Ifaprecipitatehasformedinthelysozymesolution,allowthetubetowarmatroomtemperatureandgentlyinvertthetube.Thesolutionmayremainturbidafterthisprocedure.

200ml 1XxTractorBuffer 2.5ml 50XLysozyme 400µl DNase(1unit/µl)

• TALON® xTractor Buffer (Cat.No.635625) 500ml 1XxTractorBuffer

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See Section IV for preparing buffers with theTALON® Purification Kit(Cat.No.635515)ortheTALON®BufferKit(Cat.No.635514).Ifyouhave not purchased thosekits,werecommendpreparingthefollowingbuffersforpurifyingpolyhistidine-taggedproteinsundernativeordenaturingcondi-tions.Beforepreparingotherbuffercompositions,pleaseconsultAppendixAtoevaluateresincompatibility.ForTALONMagneticBeads(seeSectionV),usethesameEquilibration/Wash Bufferaswiththeresins,andelutewithanimidazole-basedelutionbuffercontainingahigherconcentrationofimidazolethanthatusedtoelutefromtheresins,asdescribedinSectionV.D.

Choosing Buffers

Todecreasetheamountofnonspecificallyboundproteins,werecommendusingtheEquilibration/Wash BufferatpH7.0duringpurification;however,ifyourtargetproteinismorestableatpH8.0,orifitdoesnotadsorbatpH7.0,usetheEquilibrationBufferatpH8.0(inplaceoftheEquilibration/WashBuffer)duringallextractionandwashsteps.NotethatatelevatedpHvalues,aminoacidsotherthanhistidine,aswellasthepeptidebond,contributetoproteinadsorption.Thus,proteinswithoutapolyhistidinetagcanalsoadsorbtoIMACresins,whichdecreasesresincapacityandthefinalpurityofyourtargetprotein.Youmaychoosetouseeithernativeordenaturingbuffer conditions, depending on the solubility of your protein. Figure 5outlinesthepurificationprocedure.

A. Native Buffers

Nativeproteinpurificationregimensusebufferconditionsthatpreservethenative,three-dimensionalstructureandsurfacechargecharacteris-ticsofaselectedsolubleproteinduringharvestfromanexpressionhost.ThelowaffinityofTALONResinfornonpolyhistidine-taggedproteinsminimizescontaminantcarryover.Inaddition,increasingbufferionicstrengthcanminimizenonspecificinteractions.Regardlessofthecon-ditionsusedandthenatureofthepolyhistidine-taggedproteinbeingpurified,mostapplicationswillbenefitfromthepresenceof100–500mMNaClintheIMACbuffer.Inmanycases,addingglycerolorethyleneglycolneutralizesnonspecifichydrophobicinteractions.Smallamountsof nonionic detergent may also dissociate weakly bound species.

• 1X Equilibration/Wash buffer (pH7.0)* 50mMsodiumphosphate 300 mM NaCl • 1X Equilibration buffer (pH8.0)* 50 mM sodiumphosphate 300 mM NaCl

III. Additional Materials Required



III. Additional Materials Required continued

• 1X Elution buffer* – Imidazole Elution (pH7.0) – pH Elution (pH5.0)1

50mMsodiumphosphate 50mMsodiumacetate 300mMNaCl 300mMNaCl 150mMimidazole 1Preparefreshbeforeuse.

• HT 96-Well Plate Wash buffer (pH7.0)* 83mMsodiumphosphate 500mMNaCl 10mMimidazole

• TALON Magnetic Beads 1X Elution buffer* – Imidazole Elution (pH7.0)

50mMsodiumphosphate 300mMNaCl 250mMimidazole

B. Denaturing Buffers Denaturants, such as 6 M guanidinium, enhance protein solubility.

Because proteins overexpressed in prokaryotic systems are some-times insoluble,youmayneedtopurifyproteinsunderdenaturingconditions.Whenpurifyingproteinsunderdenaturingconditions,werecommendpreparingthebuffersindicatedbelow.

Inthepresenceof6Mguanidinium,the resin’s color will change from a pinkish-mauve to violet due to a change in metal ion hydration in response to the chaotrope.Afterremovalofthechaotrope,theresinwillreturntoapinkish-mauvecolor.Thechangetovioletdoesnotreflectanychangeinthephysicalorchemicalpropertiesoftheresin.Infact,thecolorchangecanbeusefulforindicatingthebufferinwhichtheresinissuspended,andforfollowingthemovementofguanidiniumthroughtheresinbed.

• 1X Equilibration/Wash Buffer (pH7.0)* 50 mM sodiumphosphate 6 M guanidine-HCl 300 mM NaCl

• 1X Equilibration Buffer (pH8.0)* 50 mM sodiumphosphate 6 M guanidine-HCl 300 mM NaCl

*SeeSambrook,AppendixB.21,oryourstandardprotocolforpreparingsodiumphosphatebuffer.




• 1X Imidazole Elution Buffer (pH7.0) 45 mM sodiumphosphate 5.4 M guanidine-HCl 270 mM NaCl 150 mM imidazole

• TALON Magnetic Beads 1X Elution Buffer* – Imidazole Elution (pH7.0)

45mMsodiumphosphate 5.4 M guanidine-HCl 270mMNaCl 250mMimidazole

C. Additional Buffers & Reagents

• MES Buffer 20mM2-(N-morpholine)-ethanesulfonicacid(MES),pH5.0 • 5X SDS PAGE sample buffer 15% β-Mercaptoethanol(β-ME) 15% SDS 50% Glycerol 1.5% Bromophenolblue

• Imidazole (Sigma,Cat.No.I0250)AlsosuitableforFPLCapplications • Bio-Rad Protein Assay (Bio-Rad,Cat.No.500-0001)

D. Additional Materials required for TALON® CellThru Resin

• CellThru2mlDisposableColumns(Cat.No.635512) • CellThru10mlDisposableColumns(Cat.No.635513)

E. Additional Materials for TALON® HT 96 Plate Vacuum Purification •QIAVac96(QIAGEN,Cat.No.19504),NucleoVac(MACHEREY-NA-

GEL,Cat.No.740630),orsimilarvacuummanifold •(Extra)Collection96-DeepWellTiterPlates(WhatmanCat.No.7701-

5200orEvergreenCat.No.240-8556-030)

Centrifugation • Centrifugewitharotorforcentrifugationofmicrotiterplates,such

astheAllegra6RCentrifuge(BeckmanCoulter)withtheGH3.8;GH3.8A;orJS4Beckmanrotors.

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT1320-1 1� Version No. PR6Z2142



F. Additional Materials for TALON® Single Step Columns •1XEquilibration/WashBuffer*(50mMsodiumphosphate,

300mMNaCl,pH7.0) •Wash-2Buffer*(50mMsodiumphosphate,300mMNaCl,

7.5mMimidazole,pH7.0) •1XElutionBuffer*(50mMsodiumphosphate,300mMNaCl,

150mMimidazole,pH7.0) •15mlscrew-captubes(5mlcolumns)or50mlscrew-captubes(20

mlcolumns),receivingtubesforfractionstorage •[Optional]BCAProteinAssayKit(Pierce,CatNo.23226)

*ThebuffersusedfortheSingleStepColumnscanbepreparedbydilutionofthebuf-fersinourTALONBufferKit(Cat.No.635514).SeeSectionIVforpreparationdetails.

G. Additional Materials for TALON® Magnetic Beads •Magneticseparator(colorlessorwhiteforbestvisibility,sincethe

beadsareblack) •1.5mland0.5mlmicrofugetubes •DNaseI

Note:AlthoughTALONxTractorBufferisincludedintheTALONMagneticBeadsBufferKit(Cat.No.635638),ifmoreisneeded,itisavailableasCat.No.635623.




Figure 5. Purification of polyhistidine-taggedproteins using TALON®Resin. TheprotocolsinthisUserManualaredesignedusingtheEquilibration/WashBufferatpH7.0.IfyourtargetproteinismorestableatpH8.0,orifitdoesnotadsorbatpH7.0,usetheEquilibrationBuffer(pH8.0)insteadoftheEquilibration/WashBufferduringtheextractionandwashsteps.*Use250mMimidazoleinsteadof150mMimidazolewhenelutingfromTALONMagneticBeads.

Native purification of soluble polyhistidine-tagged protein

Denaturing purification of insoluble polyhistidine-tagged protein

Equilibrate resin

Elute

pH elutionBuffer at pH 5.0

Imidazoleelution

Buffer at pH 7.0 + 150 mM imidazole*

Imidazoleelution

Buffer at pH 7.0+ 150 mM imidazole*,

5.4 M guanidinium

Pure native protein Pure denatured protein

Wash nonadsorbedmaterial

Wash

Apply to TALON™ resin



IV. Buffers for TALON® Purification and Buffer Kits

If you have purchased the TALON® Purification or Buffer Kits,preparebuf-fersasdescribedbelow.Todecreasetheamountofnonspecificallyboundproteins,werecommendusingtheEquilibration/Wash BufferatpH7.0duringpurification;however,ifyourtargetproteinismorestableatpH8.0,orifitdoesnotadsorbtotheresinatpH7.0,usetheEquilibrationBuffer(pH8.0)inplaceoftheEquilibration/WashBufferduringallextractionandwashsteps.NotethatatelevatedpHvalues,aminoacidsotherthanhistidine,aswellasthepeptidebond,canbeadsorbedbyTALONResins;Thus,underhighpHconditions(pH>8.0),proteinswithoutapolyhistidinetagcanbeadsorbed,decreasingresincapacityandthefinalpurityofyourtargetprotein.Note:Ifaprecipitateisobservedinthebuffers,warmthemat37°C,andstirorshaketodis-solveprecipitatepriortodilutingandusingthebuffers.

A. TALON xTractor Buffer: NopreparationnecessaryexceptoptionaladditionofDNaseIorLysozyme(seeSectionVII.A).

B. Equilibration Buffers 1.Diluteonepartofthe5XEquilibration/WashBufferor5XEquilibra-

tionBufferwithfourpartsofdeionizedwater. 2.CheckandcorrectpHifnecessary.The1XEquilibration/WashBuffer

shouldbepH7.0,whilethe1XEquilibrationBuffershouldbepH8.0.

C. Elution Buffer Diluteonepartofthe10XElutionBufferwithninepartsof1XEquilibra-

tion/WashBuffer(pH7.0)(or1XEquilibrationBuffer[pH8.0],dependingonthesolubilityofyourprotein)preparedinStepA.

D. Denaturing Conditions Add 6 M guanidinium to the Equilibration/Wash Buffer (pH 7.0), or

EquilibrationBuffer(pH8.0),andtheElutionBufferpreparedinStepsAandB,respectively.

Note: Performallstepsduringthepurificationprocedureinthepresenceof6Mguani-dinium.ProteinsamplescontaininghighguanidiniumconcentrationsformaprecipitatewhenloadedonSDSpolyacrylamidegels.Therefore,dialyzethesampleovernightinabufferedsolutioncontaining8Mureabeforeloadingitontothegel.

E. Wash Buffers • Ingeneral,usetheEquilibration/WashBufferatpH7.0towashnon-

adsorbedproteins.IftheproteinisnotstableatpH7.0,thenusetheEquilibrationBufferatpH8.0with5–10mMimidazole.

• Ifyourhostcell lineproducesunwantedmulti-histidineproteins,incorporateamorestringentwash:

Dilute10XElutionBufferineither1XEquilibration/WashBufferor1XEquilibrationBufferforafinalconcentrationof5–10mMimid-azole(1:300–1:150).



If you have purchased the TALON® Magnetic Beads Buffer Kit (Cat.No.635638),preparebuffersasdescribedbelow. A. TALON xTractor Buffer: Nopreparationnecessaryexceptoptional

additionofDNaseIorLysozyme(seeSectionVII.A).

B. Equilibration Buffers 1.Diluteonepartofthe5XEquilibration/WashBufferwithfourparts

ofdeionizedwater. 2.CheckandcorrectpHifnecessary.The1XEquilibration/WashBuffer

shouldbepH7.0.

C. Elution Buffer 1.Dilute one part of the 4X Elution buffer with three parts of 1X

EquilibrationBuffer. 2.CheckandcorrectpHifnecessary.The1XElutionBuffershouldbe

pH7.0.

D. Wash Buffers • Ingeneral,usethe1XEquilibration/WashBufferatpH7.0towash

non-adsorbedproteins. • If your host cell line produces unwanted histidine-rich proteins,

incorporate a more stringent wash with 10 mM imidazole in 1XEquilibration/WashBuffer:

Dilute4XElutionBuffer(1Mimidazole)in1XEquilibration/WashBuf-ferforafinalconcentrationof5–10mMimidazole(1:200–1:100).

V. Buffers for TALON® Magnetic Beads

E. Denaturing Conditions Add6MguanidiniumtotheEquilibration/WashBuffer(pH7.0)andthe

ElutionBufferpreparedinStepsBandC,respectively. Note: Performallstepsduringthepurificationprocedureinthepresenceof6Mguani-

dinium.ProteinsamplescontaininghighguanidiniumconcentrationsformaprecipitatewhenloadedonSDSpolyacrylamidegels.Therefore,dialyzethesampleovernightinabufferedsolutioncontaining8Mureabeforeloadingitontothegel.



A. Transformation of Host Cells with Expression Vectors Thefollowingprotocolisforchemically-inducedtransformationofE. coli competentcells.Performcontroltransformationsinparallel.

Note: UseJM109oranotherlac-induciblecelllinetoseeinductionofexpression.Fortightercontrolofexpressionlevels,useourPROTet6xHNBacterialExpressionSystem—especiallyrecommendedforexpressionofcytotoxicproteins.

1.Onice,thawatubecontaining100µlof0.5Mβ-mercaptoethanol(β-ME)andone50µltubeoffrozenE. colicompetentcellsforeachligation/transformation.

2.Dispense2µlof0.5Mβ-MEintoeachtubeofcompetentcellsandmix. 3.Dispense 2 µl of DNA directly into the mixture from Step 2. 4.Incubatetubesonicefor30min. 5.Heatshockforexactly30secina42°Cwaterbath. 6.Removetubesfromwaterbathandplaceonicefor2min. 7.Add250µlofSOCmediumtoeach tubeat roomtemperature. 8.Shake the tubes horizontally at 37°C for 1 hr at 225 rpm in

arotaryshakingincubator. 9.Spread transformation mixtures onto LB-ampicillin (50 µg/ml)

agarplates[containingX-gal(75µg/ml)andIPTG(1mM)ifblue-whiteselectionisdesired].Incubatetheplatesat37°Covernight.

B. Protein Expression 1.GrowanovernightcultureofE. colitransformedwithyourexpres-

sionplasmid.Ifyoucanisolateasufficientamountofproteinfromthisculture,proceedtoStep3aftertakinga1mlsampleforelectro-phoreticanalysis.Centrifugethesampleat1,000–3,000xgfor15minat4°C,removethesupernatant,andstorethecellpelletat–20°C.Note:Ifalarge-scalepreparationoftheproteinisrequired,proceedtoStep2.

2.Ifyouneedagreaterquantityofthetargetprotein,use20mlofovernightculturetoinoculate1Lofmedium.Incubatewithshakingforanother1–2hr,untiltheculturehasanabsorbanceof~0.6OD600.Removea1mlsampleoftheculture,centrifugeat1,000–3,000xgfor15minat4°C,removethesupernatantandstorethecellpelletat–20°Cforelectrophoreticanalysis.

3.Induceexpressionbyaddinganappropriateinducer.Forexample,thelacpromoterinthepHAT10expressionvectorcanbeinducedwith1mMIPTG.Continuetheincubationforanother3–5hr.

4.Removea1mlsampleoftheculture,centrifugeat1,000–3,000xgfor15minat4°C,removethesupernatant,andstorethecellpelletat–20°Cforelectrophoreticanalysis.

5.Proceedwithsamplepreparation(SectionVII).

VI. Transformation & Protein Expression



VII. Sample Preparation

A. TALON® xTractor Buffer Sample Preparation

ThisprocedurehasbeenoptimizedforextractionofnativeproteinsfromfreshorfrozenbacterialcellpelletusingTALONxTractorBuffer(Cat.No.635623).Thevolumesofthisextractioncanbeadjusted,aslongas20mlofxTractorBufferareusedper1gofcellpellet.

1.Add20mlofxTractorBufferto1gofbacterialcellpellet.Mixgently.Pipetthemixtureupanddowntofullyresuspendthepellet.Notes

•ForTALONHT96-WellPlate(Cat.No.635622),resuspend40–100mgofbacterialcellpelletin2mlofxTractorBuffer.

•Alog-phasecultureofE. coli(O.D.=0.6–0.8)wheninducedfor2–4hours,wouldbeexpectedtoprovide~20–40mgbacterialpelletfrom2mloftheculture.

2.[Optional]:Add40µlof1unit/µlDNaseIsolutionand200µlof50Xlysozymesolution.Notes

•DNaseIreducestheviscosityofthelysate,allowingformoreefficientremovalofcellulardebris.DNasecanbeusedwithoutlysozyme.However,ifyouareyouaretreatingcellswithlysozyme,thenyoumust treatcellswithDNaseIaswell.

•Lysozymehelpstofullydisruptbacterialwalls,andthusithasbeendemonstratedtobehighlybeneficialinextractionofhighmolecularweightproteins(>40KDa).However,lysozymeshouldbeomittedformammalianextractionproceduresaswellaswhenlysozymeinterfereswithyourprotein'sfunctionality.

3.Mixgently,pipettingupanddownseveraltimes. 4.Incubatewithgentleshakingfor10minatroomtemperature.(You

mayincubatethesolutionat4°C,ifdesired).Note: Attheendofthisincubationperiod,thereshouldbenovisibleparticles.Ifcellpellet fragmentsarepresent, resuspendthembypipettingsolutionupanddownandincubatingforadditional1–2min.

5.TheresultingcelllysatecannowbeapplieddirectlytoaTALONCellThruColumn,orthelysatesupernatantcanbeappliedtoanyotherTALONResincolumnaftercentrifugingat10,000–12,000xgfor20minat4°C.

B. Standard Sample Preparation to Isolate Native Proteins 1.Harvestthecellculturebycentrifugationat1,000–3,000xgfor15

minat4°C.Removethesupernatant.Ifyieldislow,usethemildextractionmethoddescribedinStep6,below.

2.Resuspendthecellpelletbyvortexingin2mlofchilled1XEquilibra-tion/WashBuffer(4°C)per25mlofculture≤100ml.Forcultures>1L,resuspendthepelletin1–2%oftheoriginalculturevolume.Note: You may omit Steps 3–4 if lysozyme treatment interferes with your protein’s functionality.



VII. Sample Preparation continued

3.Addlysozymetothe1XEquilibration/WashBufferforaconcentra-tionof0.75mg/ml.Toreducethechanceofintroducingproteases,usethehighestpuritylysozymeavailable.

4.Incubateatroomtemperaturefor20–30min.Note: Incubations at room temperature result in elevated proteolytic activities.Alternatively,youcanuselysozymeat4°Cwithlowerefficiency.Ifthistreatmenthydrolyzesthetargetprotein,usethemethoddescribedinStep6(below).Alter-natively,disruptthecellsbyrepeatedfreeze/thawcycles;thatis,flash-freezingthecellsuspensioninadryice-ethanolbathandthawinginchilledH2O.

5.Ifyoursampleis≤50ml,sonicateit3x10sec,witha30secpauseonicebetweeneachburst.Ifyoursampleis≥200ml,sonicateit3x30sec,witha2minpauseonicebetweeneachburst.ProceedtoStep7.Note:Excessivesonicationcandestroyproteinfunctionality.

6.[Optional]: High-yield, mild extraction method.Transferthecellstoachilledmortarandgrind1partcellswith2.5partsalumina(SigmaCat.No.A-2039)for2–3minoruntilthecompositionofthemixturebecomespaste-like.Add2mlchilled1XEquilibration/WashBuffer(4°C)per25mlculture.Note:Ifthereisahighlevelofproteolyticactivityinthecelllysate,werecommendadding1mMEDTA(finalconcentration)totheEquilibration/WashBufferinordertoinhibitmetalloproteasesduringtheextraction.BeforeapplicationofthesampletotheTALONResin,EDTAmustberemovedbygelfiltrationchromatography(PD-10,GEHealthcare)equilibratedwiththeEquilibration/WashBufferforIMAC.

7.Centrifugethecellextractat10,000–12,000xgfor20minat4°Ctopelletanyinsolublematerial.

8.Carefullytransferthesupernatanttoacleantubewithoutdisturb-ingthepellet.Thisistheclarifiedsample.

9.Reserveasmallportionoftheclarifiedsampleat4°CforSDS/PAGEanalysis.

10.Ifthisisthefirsttimeyouhavepreparedclarifiedsamplesfromcellsexpressingaparticularrecombinantprotein,werecommendthatyouestimatetheprotein’sexpressionlevelinthathoststrain.Todoso,performasmall-scalepurification,andthenanalyzeaportion by SDS/PAGE in parallel with protein standards. Onceexpressionisobserved,proceedwiththeappropriatepurificationprotocol(seeSectionVIII).

C. Standard Sample Preparation to Isolate Denatured Proteins 1.Harvest20–25mlofcellculturebycentrifugationat1,000–3,000

xgfor15minat4°C. 2.Resuspendthepelletin2mlofdenaturing 1XEquilibration/Wash

Buffer(pH7.0)per20–25mlofculture. 3.Gentlyagitateorrthesampleuntilitbecomestranslucent. 4.Centrifugethesampleat10,000–12,000xgfor20minat4°Cto

pelletanyinsolublematerial.




6.Setasideasmallportionof theclarifiedsample forSDS/PAGEanalysis.Thenproceedwiththeappropriatepurificationprotocol(seeSectionVIII).Note:Samplescontaining6Mguanidiniummustbedialyzedovernightagainstbuffercontaining8Mureabeforeloadingonagel.

D. Standard Sample Preparation for TALON® CellThru Resin

Sample Preparation to Isolate Native Proteins 1.Harvestthecellculturebycentrifugationat1,000–3,000xgfor15

minat4°C.Removethesupernatant. 2.Resuspendthecellpelletbyvortexingin2mlofchilled1XEquilibra-

tion/WashBuffer(4°C)per25mlofculture≤100ml.Forcultures>1L,resuspendthepelletin1–2%oftheoriginalculturevolume.Note: You may omit Steps 3–4 if lysozyme treatment interferes with your protein’s function.

3.Addlysozymetothe1XEquilibration/WashBufferforaconcentra-tionof0.75mg/ml.Toreducethechanceofintroducingproteases,usethehighestpuritylysozymeavailable.

4.Incubateatroomtemperaturefor20–30min.Note: Incubations at room temperature result in elevated proteolytic activities.Alternatively,youcanuselysozymeat4°Cwithlowerefficiency.Ifthistreatmenthydrolyzesthetargetprotein,usethemethoddescribedinStep6.Alternatively,disruptthecellsbyrepeatedfreeze/thawcycles;thatis,flash-freezingthecellsus-pensioninadryice-ethanolbathandthawinginchilledH2O.

5.Ifyoursampleis≤50ml,sonicateit3x10sec,withapausefor30seconicebetweeneachburst.Ifyoursampleis≥200ml,soni-cateit3x30sec,witha2minpauseonicebetweeneachburst.Note:Excessivesonicationcandestroyproteinfunctionality.

6.Storeasmallportionoftheclarifiedsampleat4°CforSDS/PAGEanalysis.

Sample Preparation to Isolate Denatured Proteins 1.Harvest20–25mlofcellculturebycentrifugationat1,000–3,000

xgfor15minat4°C. 2.Resuspendthepelletin2mlofdenaturing 1XEquilibration/Wash

Buffer(pH7.0)per20–25mlofculture. 3.Gentlyagitateorstirthesampleuntilitbecomestranslucent. 4.Setasideasmallportionof theclarifiedsample forSDS/PAGE

analysis.Thenproceedwiththeappropriatepurificationprotocol,below(seeSectionVIII).Note:Samplescontaining6Mguanidiniummustbedialyzedovernightagainstbuffercontaining8Mureabeforeloadingonagel.




E. Standard HT 96-Well Sample Preparation 1.Growcellsinappropriateformatforhigh-throughputanalysis. 2.Centrifugeifnecessaryandremovesupernatant. 3.Ifthetargetproteinsaresecretedinthemedium,utilizethesuper-

natantasastartingmaterialandproceedtoStep6.Ifthetargetproteinsareintracellular,proceedtothenextstep.

4.Disruptthecellsinpresenceof1XEquilibration/WashBuffer(use2mlofbufferper200mgofcellsperpurificationwell).Note:[Optional]UseTALONxTractorbufferforbetterextractionefficiency.

5.Centrifugeextractsandcollect thesupernatant tobeusedasastartingmaterial.

6.Remove50µlofeachsampleforproteinconcentrationanalyses.

F. Standard Sample Preparation for TALON Magnetic Beads When purifying proteins, more effective binding is achieved when

running clarified lysates. For screening of expression levels, crudelysatesderivedfromovernightculturescanbeaddeddirectlytothebeads(seeSectionVII.G).

Sample Preparation to Isolate Native Proteins 1.Harvestthecellculturebycentrifugationat1,000–3,000xgfor15

minat4°C.Removethesupernatant.Thepelletcaneitherbeusedimmediatelyorstoredfrozenat–70°C.

2.Add0.5mlofxTractorBufferper25mgofcellpellet.ThevolumeofxTractorBuffercanbeincreasedordecreaseddependingonthesizeofthecellpellet.

3.[Optional]: Add1µlof1unit/mlDNaseIsolution. 4.Mixgently,pipetingupanddownseveraltimes. 5.Incubatewithgentleshakingfor10minatroomtemperature,or

at4°C,ifdesired. 6.[Optional]: High-yield, mild extraction method.Transferthecells

toachilledmortarandgrind1partcellswith2.5partsalumina(SigmaCat.No.A-2039)for2–3minoruntilthecompositionofthemixturebecomespaste-like.Add1mlchilled1XEquilibration/WashBuffer(4°C)per25mgofcellpellet.

7.Centrifugeat10,000-12,000xgfor20minat4°C.Note:TheuncentrifugedcrudecelllysatecanalsobeappliedtoTALONMagneticBeads.However,thelysatemayhavetobedilutedfurtherorrequiremoreDNasetopreventthebeadsfromfailingtomigratetothemagnetbecauseofthehighviscosityofthesolution.






9.Reserveasmallportionof theclarifiedsampleat4°CoroniceforproteinassaysandSDS-PAGEanalysis.ThenproceedwiththeTALONMagneticBeadspurificationprotocol(SectionVIII.I).

Sample Preparation to Isolate Denatured Proteins 1.Harvestthecellculturebycentrifugationat1,000–3,000xgfor15

minat4°C.Removethesupernatant.Thepelletcaneitherbeusedimmediatelyorstoredfrozenat–70°C.

2.Add0.5mlofdenaturing 1XEquilibration/WashBufferper25mgofcellpellet.Thevolumeofthebuffercanbeincreasedordecreaseddependingonthesizeofthecellpellet.

3.Gentlyagitateorstirthesampleuntilitbecomestranslucent. 4.Centrifugethesampleat10,000–12,000xgfor20minat4°Cto

removeanyinsolublematerial. 4.Carefullytransferthesupernatanttoacleantubewithoutdisturb-

ingthepellet.Thisistheclarifiedsample. 5.Setasideasmallportionoftheclarifiedsampleat4°Coronice

forproteinassaysandSDS-PAGEanalysis.ThenproceedwiththeTALONMagneticBeadspurificationprotocol(SectionVIII.I).Note: Samplescontaining6Mguanidinemustbedialyzedovernightagainstbuffercontaining8Mureabeforeloadingonagel.

G. Sample Preparation Directly from Overnight Cultures for TALON Magnetic Beads

Ifscreeningtransformantsforexpressionlevels,pickasinglecolonyfromtheplateandinoculate4.5mlmedium.Incubatethecultureat37°CuntiltheOD600reaches~0.6–0.8AU(mid-logphase).Theninduceproteinexpressionwiththerecommendedconcentrationofinduceragent(dependingonyourexpressionstrainandtheexpressionplas-midbeingused).Continuetogrowtheculturewithrigorousshakingat37°Cforanother4hrorovernight.Alternatively,followyourstandardinductionorexpressionprotocol.

1.Diluteovernightculture1:1withxTractorBufferandaddDNasetoaconcentrationof1unit/mlofculture.(Forexample,dilute0.5mlofanovernightculturewith0.5mlofxTractorBufferandadd1unitofDNase.)

2.Mixthoroughlyat4°Cfor30min. 3.[Optional] Ifthecultureisstilltooviscous,diluteitwithsufficient

5X Equilibration/Wash Buffer to obtain a final concentration of1XEquilibration/WashBuffer.

4.CheckpHtoensureitfallsbetween7–8foroptimalbindingandproceed with theTALON Magnetic Beads purification protocol(SectionVIII.I).



PLEASE READ ENTIRE PROTOCOL BEFORE STARTING.

A. General Information 1.Performallmanipulationsat4–8°Cinordertomaintainprotein

stabilityandimproveyield. 2.This protocol is designed using the Equilibration/Wash Buffer

(pH 7.0).IfyourtargetproteinismorestableatpH8.0,orifitdoesnotadsorbatpH7.0,usetheEquilibrationBufferatpH8.0(insteadoftheEquilibration/WashBuffer)duringextractionandwashsteps.

3.Areducingagent,suchas10mMβ-ME,oraproteaseinhibitor,suchasPMSF,intheEquilibration/WashBuffer(pH7.0),mayimprovethestructuralstabilityoffragileproteinsduringsamplepreparation.SeeAppendixAforcompatibilityinformation.Note:Dependingontheconcentrationandvolumeoftheadditiveyouwishtouse,youmayneedtoremakethebufferstopreservetherecommendedconcentrationofNaClandbufferingagent. DTT and DTE are not compatible with this TALON protocol in any concentration.

4.If thecell lysatecontainsahighlevelofproteolyticactivity,werecommendadding1mMEDTAtotheEquilibration/WashBuffer(pH7.0)toinhibitmetalloproteasesduringtheextraction. However, before applying the sample to the resin, remove EDTA using a gel filtration column (such as PD-10, GE Healthcare) equilibrated with the Equilibration/Wash Buffer.Insomecases,thehostcellproduceslowmolecularweightchelatorsthatcanalsoberemovedusinggelfiltration.

Chelators can be detected by applying your sample to a smallcolumnpackedwithTALONResin.Ifthetopofthecolumnlosesitscharacteristicpinkcolor,andthecolorlessfrontmovesinthedirec-tionoftheflow,orifyouobtainpinkfractionsduringbatchadsorp-tion,youmustequilibratethesampleusingagelfiltrationcolumn.

5.Overexpressedrecombinantproteinscanaccumulateininsolubleinclusionbodies.Inordertodetermineoptimalextraction/purifica-tionconditions,youmustdeterminethedistributionoftheproteininsolubleandinsolubleforms.PerformapreliminarySDS/PAGEanalysis of protein extracts obtained under native conditions,followedbyextractionoftheresidualproteinsunderdenaturingconditions.Takecaretousethesameextractionvolumesforbothnativeanddenaturingextracts,andrunthecellextractbeforein-ductionasacontrolinonelanetoidentifythetargetprotein.Useofdenaturingconditions isrecommendedonly if thebiologicalactivityofthetargetproteinwouldnotbeaffectedbydenaturationorisunimportant.Itispreferabletousenativeconditionsforex-tractionevenifonly5–10%ofthetargetproteinissoluble.

VIII. Protein Purification Protocols



6.Thebuffervolumesinthefollowingprotocolswereoptimizedforpurifying theHAT-DHFR fusionprotein from20–25mlofE. coliculture.Dependingontheexpressionlevelandanticipatedyield,youmayneedtoadjustthebuffervolumesforotherproteins.Asastartingpoint,use2mlofbufferper20–25mlofculture.

7.Ifyouarepurifyingproteinfromharvestedeukaryoticcells,lysethecellsinanappropriatebuffercontainingamilddetergent(Sambrook&Russell,2001).SeeAppendixAforcompatiblebufferadditives.Note that EDTA and EGTA are not compatible with these protocols be-cause these metal-chelating reagents strip the cobalt from the resin.

8.Carefullycheckthesample’sappearanceafterlysisorsonication.BacterialsamplesoftenremainviscousfromincompleteshearingofgenomicDNA.CompleteDNAfragmentationimprovesproteinyieldsandallowsefficientremovalofcellulardebrisduringcentrifu-gation.Youmaydecreasethesample’sviscositybydigestionfor20–30minatroomtemperaturewith2.5µg/mlofDNaseI.Remem-berthatproteolyticactivityismuchhigheratroomtemperature.Alternatively,dilutethesamplefivefoldwithEquilibration/WashBufferbeforeapplyingittotheresin.Thisprocedureshouldnotsignificantlyaffectrecovery.

Notes on Protein Purification methods using TALON® Resin Thefollowinggeneralguidelinesareusedforpurifyingpolyhistidine-

taggedproteinfromtransformedE. colicultures.TableIprovidesanoverviewofTALON®Resinproteinpurificationmethodsandapplica-tions.Chooseamethodthatbestsuitsyourresearchneeds.

• Use2mlofresinsuspensionper~3mgofanticipatedpolyhisti-dine-tagged protein. 2 ml of homogeneously resuspended resinwillprovide1ml(bedvolume)ofTALONResin.

• Thebuffersandpurificationconditionsshouldworkwellformostsoluble,monomericproteinsexpressedinE. coli.

• Initially,testeachdifferentexpressionsystemandpolyhistidine-taggedproteininsmall-scalebatchpurificationtodetermineexpressionlevelsandtooptimizetheprotocol.Amini-scalebatchpurificationprotocolisprovidedinAppendixB;alternatively,youcanuseaTALONspincolumn.

• Purificationmethodsthatworkwithnickelorzinc-basedIMACresinsshouldalsoworkwiththeseresins.However,someoptimizationmayberequired.

TALON® CellThru Considerations Theprocedureforpurifyingpolyhistidine-taggedproteinsusingTALON

CellThruResinisessentiallythesameasotherTALONResinswiththefollowingsignificantdifferences.

VIII. Protein Purification Protocols continued



1. Extracellular Proteins Iftherearenochelatingagentsinthefermentationliquidandthe

pHis≥7.0,youcanapplysampledirectlyontoaprepackedcolumn.Otherwise, a desalting/equilibration step is necessary (such asultrafiltrationorgelfiltrationwithSephadexG25).

2. Intracellular Proteins Forpurifying intracellularproteins, apply the sonicatedsample

containingyourtargetproteins,directlyontoaprepackedcolumn.Thereisnoneedforcentrifugation.Electrophoresiswillrevealthatsomeofthetargetproteinhaspassedthroughthecolumnwithoutadsorption.Toalargeextentthematerialpassingthroughthecol-umnisinsolubleprotein,whichwouldnormallyhavebeenremovedduring high-speed centrifugation.The amount of non-adsorbedtargetproteinwillalsovaryasafunctionofsonicationefficiency.

3. Chromatography Considerations TALONCellThruBeadshaveadiameterof300–500µm;therefore,

useacolumnwithafilterporesizeof90–130µmtoadequatelypasscellulardebris.WerecommendusingourCellThru2ml&10mlDis-posableColumns(Cat.No.635512&635513).The2mlcolumnsaresuitablefor1–2mlbedvolumes,whilethe10mlcolumnsaresuitablefor5–10mlbedvolumes.Becausethecolumnfiltershavealargerporesizeandpermithigherflowrates,youmayneedtoincubateyoursamplewiththeresinfor5minbeforelettingitflowthrough.Ifnecessary,passthesamplethroughthecolumnasecondtime.

Thetechniqueofexpandedbedchromatographyworkswellwiththeseresinsasthematerialcanflowthroughtheresinmoreef-fectively.Flowratesmayhavetobeadjustedtogetthemaximumbindingefficiencywhenusingthistechnique.

B. Batch/Gravity-Flow Column Purification

For column IMAC usingTALON Resins, we recommend a hybridbatch/gravity-flowprocedure.Thismethodcombinesthespeedandconvenienceofabatchprocedurewiththeexceptionallyhighpurityofthegravity-flowcolumnmethod.Inthishybridprocedure,thebind-ingandinitialwashingstepsareperformedinabatchformattosavetime,eliminateextraneousdebris,andavoidcolumnclogging.Aftertheinitialwashes,theresinistransferredtoacolumnforadditionalwashingandproteinelution.

1.ThoroughlyresuspendtheTALONResin. 2.Immediatelytransfertherequiredamountofresinsuspensiontoa

steriletubethatwillaccommodate10–20timestheresinbedvolume. 3.Centrifugeat700xgfor2mintopellettheresin.




4.Removeanddiscardthesupernatant. 5.Add10bedvolumesof1XEquilibration/WashBufferandmixbriefly

topre-equilibratetheresin. 6.Recentrifugeat700xgfor2mintopellettheresin.Discardthe

supernatant. 7.RepeatSteps5and6. 8.AddtheclarifiedsamplefromSectionVI.A,B,orCtotheresin. 9.Gentlyagitateatroomtemperaturefor20minonaplatformshaker

toallowthepolyhistidine-taggedproteintobindtheresin. 10.Centrifugeat700xgfor5min. 11.Carefullyremoveasmuchsupernatantaspossiblewithoutdisturb-

ingtheresinpellet. 12.Washtheresinbyadding10–20bedvolumesof1XEquilibration/

WashBuffer.Gentlyagitatethesuspensionatroomtemperaturefor10minonaplatformshakertopromotethoroughwashing.

13.Centrifugeat700xgfor5min. 14.Removeanddiscardthesupernatant. 15.RepeatSteps12–14. 16.Addonebedvolumeofthe1XEquilibration/WashBuffertothe

resin,andresuspendbyvortexing. 17.Transfertheresintoa2mlgravity-flowcolumnwithanend-cap

inplace,andallowtheresintosettleoutofsuspension. 18.Removetheend-cap,andallowthebuffertodrainuntilitreaches

thetopoftheresinbed,makingsurenoairbubblesaretrappedintheresinbed.

19.Washcolumnoncewith5bedvolumesof1XEquilibration/WashBuffer.

20.[Optional]:Ifnecessary,repeatStep19undermorestringentcon-ditionsusing5–10mMimidazolein1XEquilibration/WashBuffer(SectionIV.D).

21.Elutethepolyhistidine-taggedproteinbyadding5bedvolumesofElutionBuffertothecolumn.Collecttheeluatein500µlfractions.Note: Undermostconditions,themajorityofthepolyhistidine-taggedproteinwillberecoveredinthefirsttwobedvolumes.

22.UsespectrophotometricandSDS/PAGEanalysestodeterminewhichfraction(s)contain(s)thebulkofthepolyhistidine-taggedprotein.Note:UseaBradfordproteinassay(Bradford,1976)orUVabsorbanceat280nm.UseUVabsorbanceonlyifyouareelutingsufficientproteintoexceedtheabsorbanceoftheimidazoleat280nm.Alternatively,dialyzethefractionsovernightagainsttheEquilibration/WashBuffer,andthenmeasuretheirUVabsorbanceat280nm.




C. Large-Scale Batch Purification Thismethodpurifiespolyhistidine-taggedproteinsfasterthangravity-

flowcolumns;however,batchwashesremoveimpuritieslessefficientlythangravity-flowcolumns.Therefore,theyrequirelargerwashbuffervolumestoobtainpurepolyhistidine-taggedproteins.

1.ThoroughlyresuspendTALONResin. 2.Transferrequiredamountofresintoaglassfilterwithaporesizeof

10–20µm. 3.Applyavacuumtothefiltertoremoveexcessethanol. 4.Add 5 bed volumes of deionized water to the resin, and apply

vacuum. 5.Add5bedvolumesof1XEquilibration/WashBuffertotheresin,

andapplyvacuum. 6.RepeatStep5twotimes. 7.Addcrudelysate(CellThruResin)orclarifiedsample(otherthan

CellThruResin)totheresin,andmixfor3–5min. 8.Applyvacuumandcollectthefiltrate. 9.Washtheresinbyadding10–20bedvolumesof1XEquilibration/

WashBuffer.Gentlyagitatethesuspensionatroomtemperaturefor10minonaplatformshakertopromotethoroughwashing.

10.Applyvacuumtoremovebuffer. 11.Repeattheabovewash(Steps9–10)2–3times. 12.[Optional]: If necessary, repeat Step 11 under more stringent

conditionsusing5mMimidazolein1XEquilibration/WashBuffer(SectionIV.D).

13.Elutethepolyhistidine-taggedproteinbyadding5bedvolumesofElutionBuffer.

14.Gentlyagitatesuspensionatroomtemperaturefor5min. 15.Apply vacuum, and collect the purified polyhistidine-tagged

protein. 16.RepeatSteps13–15twotimes,collectingseparatefractions. 17.Use spectrophotometric and SDS/PAGE analyses to determine

whichfraction(s)contain(s)themajorityofthepolyhistidine-taggedprotein.Note:Samplescontaining6Mguanidiniummustbedialyzedovernightagainstbuffercontaining8Mureabeforeloadingonagel.




D. Medium-Pressure Column Purification 1.Assemblecolumnaccordingtothemanufacturer’sinstructions. 2.ThoroughlyresuspendTALONSuperflowResin.Slowlypourthe

slurryintothecolumn,andavoidintroducingairbubbles. 3.Allowresintosettle.Acceleratethisprocessbyallowingthebuffer

toflowthroughthecolumnwithaperistalticpumpattachedtotheoutputofthecolumn.Donotexceedaflowrateof5ml/min/cm2.Donotallowtheresintodryout.Ifthisoccurs,resuspendtheresinandrepackthecolumn.

4.Insertandadjustthetopadaptorandconnectthecolumntothechromatographysystemaccordingtomanufacturer’sinstructions.Note:Avoidtrappingairbetweentheadaptorandtheresinsurface.

5.Equilibratethecolumnwith1XEquilibration/WashBuffer.Donotexceeda5ml/min/cm2flowrate.Monitortheeluantat280nm;thebaselineshouldbestableafterwashingwith5–10columnvolumes.

6.Applytheclarifiedsampletothecolumnafterfilteringitthrougha0.22-µmfilterandwashwithEquilibration/WashBufferuntilthebaseline(280nm)isstable.Monitorcolumnbackpressureduringsampleapplication.Startcollectingfractions.

Note:Ifthesampleisveryviscous,thecolumnpressuremayexceedtherecom-mendedvalue(150psi,1.0MPa).Reducetheflowrateordilutethesampletobringthepressureintoanacceptablerange.

Loadthesampleataflowrateof0.5–1.0ml/min/cm2toensurethatthepolyhistidine-taggedproteinwillbindtotheresin.Iftheproteindoesnotbind,reducetheflowratefurther.Ifdesired,increasetheflowrateforwashingandproteinelution.

Ifthetargetproteinisunstableatroomtemperature,performthechro-matographyat4°C.Alternatively,useflowratesupto5ml/min/cm2toload,wash,andelutetheprotein.Capacitywilldecreaseby10–15%,butonaverage,achromatographyrunshouldonlytake15–20min.

7.Washcolumnwith10–20column-volumesofEquilibration/WashBuffer,oruntilthebaselineat280nmisstable.Ifnecessary,add5–10mMimidazoletotheEquilibration/WashBuffer.

8.Elutethepolyhistidine-taggedproteinwith5–10column-volumesofElutionBuffer.Thepolyhistidine-taggedproteinusuallyelutesinthesecondandthirdcolumnvolumes.

9.UsespectrophotometricandSDS/PAGEanalysestodeterminewhichfraction(s)contain(s)themajorityofthepolyhistidine-taggedprotein.Note:Samplescontaining6Mguanidiniummustbedialyzedovernightagainstbuffercontaining8Mureabeforeloadingonagel.

10.Ifyouplantostore,regenerate,andreusearesin-packedcolumn,seeSectionIX.C.




E. 5 ml TALON® Single Step Column PurificationTheseprotocolsaredesignedforusewithTALONSingleStepColumnsforgravityfloworcentrifugepurification.• Forpurificationsoflessthan10samples,werecommendusingthe gravityflowprocedure.Forhigh-throughputpurificationofmorethan 10samples,thecentrifugeprocedureshouldbefaster.• Thebuffersusedinthisprocedurecanbeeasilypreparedbydilution

fromthestockbuffersfromourTALONBufferKit(Cat.No.635514).SeeSectionIVforpreparationdetails.

1.Samplepreparationandlysis

a.TALONSingleStepColumnscanbeusedforpurificationofanyhistidine-taggedproteinfromanE. coliculture.Forexample,ifscreeningtransformantsforexpressionlevels,pickasinglecolonyfromtheplateandinoculate4.5mlmedium.Incubatethecultureat37°CuntiltheOD600reaches~0.6–0.8AU(mid-logphase).Theninduceproteinexpressionwiththerecommendedconcentrationofinduceragent(dependingonyourexpressionstrain and the expression plasmid being used). Continue togrowtheculturewithrigorousshakingat37°Cforanother4hrorovernight.Alternatively, followyourstandardinductionorexpressionprotocol.[Optional:remove200µloftheexpressioncultureforSDS/PAGEanalysis.]

b.PlacethebottomclosurefirmlyonaTALONSingleStepCol-umn,removethetopcap,add4.5mlcultureandthenreplacethetopcap.Mixthesuspensioneitheronacarouselshakerfor20–30minatroomtemperatureorbyinvertingthetubeevery2minforatotalof30min.

c.Remove the top cap and the bottom closure and place theTALONSingleStepColumnintoaReceivingTube.Proceedwitheitherthegravityfloworthecentrifugeprocedure.

2.GravityFlowProcedure a.Lettheextractdrainbygravityflow.Removethecolumnfromthe

receivingtubeandreplacethebottomclosure.[Optional:remove200µlofnon-adsorbedmaterial fromtheReceivingTubeforSDS/PAGEandProteinAssayanalysis(Step4).]

b.Add4.5ml1XEquilibration/WashBuffer,replacethetopcapandresuspendtheresinbyinvertingthecolumn.RemovethebottomclosureandputthecolumnintoaReceivingTube.Allowthewashtodrainbygravityflow.[Optional:remove200µlofWash-1fromtheReceivingTubeforSDS/PAGEandProteinAssayanalysis.]

c.[Optional] Forimprovedpurityoftargetprotein,repeatStepbtwice.




d.Replace the bottom closure, then add 4.5 mlWash-2 Buffer.Replacethetopcapandresuspendtheresinbyinvertingthecolumn. Remove the bottom closure and put column into aReceivingTube.Letthebufferdrainbygravityflow. [Optional:remove200µlofWash-2fromtheReceivingTubeforSDS/PAGEandProteinAssayanalysis.]

e.[Optional]Forimprovedpurityofthetargetprotein,repeatthewashinStepdtwice.

f.Add1.0mlElutionBufferandresuspendtheresinbyinvertingthecolumnsfor2min.

g.RemovethebottomclosureandputthecolumnintoaReceivingTube.Allowtheelutionfractiontodrainbygravityflow.ProceedwithStep4.ProteinAnalysis.

3.CentrifugeProcedure a.Centrifugeat700xgfor2min.TakethecolumnfromtheRe-

ceivingTubeandreplacethebottomclosure.[Optional:remove200µlofnon-adsorbedmaterialcollectedintheReceivingTubeforSDS/PAGEandProteinAssayanalysis.]

b.Add4.5ml1XEquilibration/WashBuffer,replacethetopcapandresuspendtheresinbyinvertingthecolumn.RemovethebottomclosureandputthecolumnintoaReceivingTube.Centrifugeat700xgfor2min.RemovethecolumnfromtheReceivingTubeand replace thebottomclosure. [Optional: remove200µlofWash-1forSDS/PAGEandProteinAssayanalysis.]

c.[Optional] Forimprovedpurityoftargetprotein,repeatStepbtwice.

d.Add4.5mlWash-2Buffer,replacethetopcapandresuspendtheresinbyinvertingthecolumn.RemovethebottomclosureandputtheSingleStepColumnintoaReceivingTube.Centrifugeat700xgfor2min.RemovethecolumnfromtheReceivingTubeand replace thebottomclosure. [Optional: remove200µlofWash-2fromtheReceivingTubeforSDS/PAGEandProteinAs-sayanalysis.]


f.Add1mlElutionBufferandresuspendtheresinbyinvertingthecolumnfor2min.

g.RemovethebottomclosureandputtheTALON®SingleStepColumnintoaReceivingTube.Centrifugethecolumninthetubeat700xgfor2min.ProceedwithStep4.ProteinAnalysis.





4.ProteinAnalysis Determineamountofproteinina1:10dilutionofthenon-adsorbed

fractionsandtheamountofproteininthe(undiluted)elutionfractionbyperformingaBio-RadProteinAssay.AnalyzethesamplesbySDS/PAGEtodeterminethepurityofthetargetprotein(Elutionfraction).Note:ABCAProteinAssay (seeSection III) canbeperformedonanundilutedsampleofthenon-adsorbedfraction,ifdesired.

F. 20 ml TALON® Single Step Column PurificationTheseprotocolsaredesignedforusewiththe20mlTALONSingleStepColumnsforgravityfloworcentrifugepurification.• Forpurificationsoflessthan10samples,werecommendusingthegravityflowprocedure.Forhigh-throughputpurificationofmorethan10samples,thecentrifugeprocedureshouldbefaster.• Thebuffersusedinthisprocedurecanbeeasilypreparedbydilution

fromthestockbuffersfromourTALONBufferKit(Cat.No.635514).SeeSectionIVforpreparationdetails.

1.Samplepreparationandlysis

a.TALONSingleStepColumnscanbeusedforpurificationofanyhistidine-taggedproteinfromanE. coliculture.Forexample,ifscreeningtransformantsforexpressionlevels,pickasinglecolonyfromtheplateandinoculate25mlmedium. Incubatethecultureat37°CuntiltheOD600reaches~0.6–0.8AU(mid-logphase).Theninduceproteinexpressionwiththerecommendedconcentrationofinduceragent(dependingonyourexpressionstrain and the expression plasmid being used). Continue togrowtheculturewithrigorousshakingat37°Cforanother4–6hrorovernight.Alternatively,followyourstandardinductionorexpressionprotocol. [Optional: remove200µloftheexpressioncultureforSDS/PAGEanalysis.]

b.EnsurethattheendcapisfirmlyontheTALONSingleStepCol-umn,removethetopcap,add20mlcultureandthenreplacethetopcap.Mixthesuspensioneitheronacarouselshakerfor20–30minatroomtemperatureorbyinvertingthetubeevery2minforatotalof30min.

c.Remove the top cap and the end cap and place theTALONSingleStepColumnbackintotheReceivingTube.Proceedwitheitherthegravityfloworthecentrifugeprocedure.

2.GravityFlowProcedure a.Lettheextractdrainbygravityflow.Removethecolumnfrom

thereceivingtubeandreplacetheendcap.[Optional:remove200µlofnon-adsorbedmaterial fromtheReceivingTubefor




SDS/PAGEandProteinAssayanalysis(Step4).] b.Add20ml1XEquilibration/WashBuffer,putthecolumninto

afreshReceivingTube,replacethetopcapandresuspendtheresinbyinvertingthecolumn.RemovetheendcapandputthecolumnbackintotheReceivingTube.Allowthewashtodrainbygravityflow.[Optional:remove200µlofWash-1fromtheReceivingTubeforSDS/PAGEandProteinAssayanalysis.]

c.[Optional] Forimprovedpurityoftargetprotein,repeatStepbtwice. d.Replacetheendcap,putthecolumnintoafreshreceivingtube,

thenadd20mlWash-2Buffer.Replacethetopcapandresus-pendtheresinbyinvertingthecolumn.RemovetheendcapandreplacethecolumnintotheReceivingTube.Letthebufferdrainbygravityflow.[Optional:remove200µlofWash-2fromtheReceivingTubeforSDS/PAGEandProteinAssayanalysis.]

e.[Optional] Forimprovedpurityofthetargetprotein,repeatthewashinStepdtwice.

f.Replacetheendcap,add2.0mlElutionBuffer,placethe20mlcolumnintoafreshreceivingtube,andresuspendtheresinbyinvertingthecolumnfor2min.Foranadditional10–15%ofpuri-fiedprotein,repeatelutionwithanadditional2.0mlElutionBuffer.

g.RemovetheendcapandreplacethecolumnintotheReceivingTube.Allowtheelutionfractiontodrainbygravityflow.ProceedwithStep4.ProteinAnalysis.

3.CentrifugeProcedure a.Centrifugethecolumnat700xgfor2min.Takethecolumnfrom

theReceivingTubeandreplacetheendcap.[Optional:remove200µlofnon-adsorbedmaterialcollectedintheReceivingTubeforSDS/PAGEandProteinAssayanalysis.]

b.Placethecolumninafreshreceivingtube,thenadd20mlof1XEquilibration/WashBuffer,replacethetopcapandresuspendthe resin by inverting the column. Remove the end cap andreplacethecolumnintotheReceivingTube.Centrifugeat700xgfor2min.[Optional:remove200µlofWash-1forSDS/PAGEandProteinAssayanalysis.]

c.[Optional] Forimprovedpurityoftargetprotein,repeatStepbtwice. d.RemovethecolumnfromtheReceivingTubeandreplacethe

endcap.Placethecolumninafreshreceivingtube,add20mlWash-2Buffer,replacethetopcapandresuspendtheresinbyinvertingthecolumn.RemovetheendcapandreplacetheSingleStepColumnintotheReceivingTube.Centrifugeat700xgfor2min.[Optional:remove200µlofWash-2fromtheReceivingTubeforSDS/PAGEandProteinAssayanalysis.]





f.RemovethecolumnfromtheReceivingTubeandreplacetheendcap.Add2mlElutionBuffer,placethecolumninafreshReceiv-ingtube,closethetopcapandresuspendtheresinbyinvert-ingthecolumnfor2min.Foranadditional10–15%ofpurifiedprotein,repeatelutionwithanadditional2.0mlElutionBuffer.

g.RemovetheendcapandreplacetheTALONSingleStepColumnintotheReceivingTube.Centrifugethecolumninthetubeat700xgfor2min.ProceedwithStep4.ProteinAnalysis.

4.ProteinAnalysis Determineamountofproteinina1:10dilutionofthenon-adsorbed

fractionsandtheamountofproteininthe(undiluted)elutionfractionbyperformingaBio-RadProteinAssay.AnalyzethesamplesbySDS/PAGEtodeterminethepurityofthetargetprotein(Elutionfraction).Note:ABCAProteinAssay (seeSection III) canbeperformedonanundilutedsampleofthenon-adsorbedfraction,ifdesired.

G. TALONspin™ Column Purification Important Points • Beforeproceedingwithpurification,determinetheconcentrationof

polyhistidine-taggedproteininyoursampleusingthemini-batchscreeningprotocol(AppendixB).Alternatively,runasampleoftheclarifiedlysatedirectlyonSDS/PAGE,andestimatetheamountofpolyhistidine-taggedproteinbybandintensity.

• Avoidexcessivelyconcentratedorviscouslysates. SeeTroubleshoot-ing(SectionIX.B.2)fortipsonreducingsampleviscosity.

• Iftheconcentrationofpolyhistidine-taggedproteininthelysateisverydilute,useonecolumntoenrichtheproteinfromseveral0.6–1mllysatealiquots.SimplyrepeatSteps11–16(below)untilthedesiredamountoflysatehasbeenprocessed.Alternatively,concentratethepolyhistidine-taggedproteinbyreducingthesamplevolume.

• Thecentrifugationrotorandspeedmayaffectyourresults.Ideally,youshouldcentrifugeTALONspinColumns inaswingingbucketrotor to allow the sample to pass through the resin uniformly.However,afixedanglerotororamicrocentrifugeisalsoacceptable.Centrifugationspeedshigherthan700xgmaycauseirregularitiesintheflowofsolutionthroughtheresinbed,andthus,decreasetheperformanceofthecolumn.

1.HoldtheTALONspinColumnuprightandflickituntilallresinfallstothebottomofthecolumn.Then,snapoffthebreakawayseal.Note:Savewhiteend-capforlateruse.

2.Placecolumninthe2mlmicrocentrifugetube.




3.Removethecleartop-capandcentrifugecolumnat700xgfor2mintoremovethestoragebufferfromtheresinbed.Note:Theresinbedwillappearsemi-dryaftercentrifugation.

4.Removecolumnfromcentrifuge,andplacethewhiteend-capoverthemaleluerfitting.

5.Add 1 ml 1X Equilibration/Wash Buffer and mix briefly to pre-equilibratetheresin.

6.Recentrifugeat700xgfor2mintopellettheresin.Discardthesupernatant.

7.RepeatSteps7and8,twice. 8.AddtheclarifiedsamplefromSectionVII.A,BorCtotheresin. 9.Add0.6–1mlofsampletothecolumn,andreplacethecleartop-cap. 10.Allowsampletopassivelywettheresinbedfor30sec. 11.Mixorvortexcontentsbrisklyfor1–2sec,completelyresuspending

theresininthelysate. 14.Gentlyagitate thesuspension for5min toallowpolyhistidine-

taggedproteinbinding.Do not vortex. 15.Removebothcapsfromcolumnandplacecolumninsidethe2ml

microcentrifugetube. 16.Centrifugeat700xgfor2min. 17.Removethecolumnandmicrocentrifugetubefromthecentrifugerotor,

makingsurethatallofthesamplehaspassedthroughtheresinbed.Note:Viscoussamplesmayrequireadditionalcentrifugation.

18.Savethe2mltube,butdiscardtheflowthrough. 19.Placemicrocentrifugetubeinrotor. 20.Placewhiteend-caponthecolumn,andadd1mlof1XEquilibra-

tion/WashBuffer.Closethecolumnwiththecleartop-cap. 21.Allowthebuffertopassivelywettheresinbedfor30sec. 22.Agitateorvortexbrisklyforafewsecuntiltheresiniscompletely

resuspended. 23.Gentlyagitatefor5min. 24.Removebothcaps,andcentrifugeat700xgfor2min. 25.Repeat Steps 18–24. Repeat twice for particularly concentrated

lysates,orifnecessary,toimprovepurity. 26.Examinetheresinbedtoensurethatitappearssemi-dry,andto

ensurethatallwashbufferhasdrainedfromtheresinbedandthecolumnend.

27.Discardtheused2mlmicrocentrifugetube. 28.Ifnecessary,repeatthespintoremovealltracesofwashbuffer.




29.Replacethewhiteend-caponthespincolumn. 30.Add400–600µlofElutionBuffer.

Note: Alternatively,use100mMEDTA(pH8.0)ifitdoesnotinterferewithdownstreamapplicationsoftheprotein.SampleselutedwithEDTAwillalsocontaincobalt.

31.Allow1minforElutionBuffertopassivelywettheresinbed. 32.Brieflyagitateorvortextoresuspendtheresin. 33.Placeafresh2mlcollectiontubeintocentrifugerotor. 34.Removebothcapsandplacecolumnintothe2mlcollectiontube. 35.Centrifugesampleat700xgfor2min. 36.RepeatSteps30–35.

Note:The polyhistidine-tagged protein sample can generally be recovered in800–1,200µlofElutionBuffer,butitmaybenecessarytousealargerElutionBuffervolumeorrepeatSteps30–35.

37.Determinepolyhistidine-taggedproteinyieldusinggelorspectro-photometricanalysis.Note:Ifthepurityofthepolyhistidine-taggedproteinpreparationisunsatisfactory,refertotheprocedureintheTroubleshootingGuideSectionX.C.2.

H. TALON® HT 96-Well Purification ProtocolEachwelloftheTALONHT96-WellPlatehasacapacityofupto1.0mgofpolyhistidinetaggedprotein.Inordertoobtainthemaximumyieldofpureprotein,donotattempttoloadmorethan1.0mgofpolyhisti-dine-taggedprotein/well.Also,observethefollowingguidelines:

•Whenusingpipettetipstomixtheresin,usewide-boretips,orcutthe tips tomake theopeningwider.Thiswill reducemechanicaldamagetoproteinsaswellasresin.

•TALONResinisdesignedtopermitbuffertoflowthroughfreely.Therefore,whentheHT96-WellPlateisnotonthevacuummanifoldoroveraCollectionPlate,werecommendthatitiskeptontheBaseSealthatactsasatemporarystopper.

•Theamountofsampleappliedtoawellshouldnotexceedtheca-pacityof1.0mg/well.

•Avoidoverdryingtheresinunderthevacuum.Forthebestresults,keeptheresinwet.

•Whenusingvacuummanifold,adjustthevacuumtoobtainaflowrateof1–2dropspersec(~100–200mmHgor2–4psi).

1.Unpackingandremovalofseals HT96-Wellplatescomewithsolidplatesealstopreventresinfrom

leakingduringtransportation.Beforeremovingtheupperseal,werecommendperforminga2mincentrifugationstepat500xgtopack resinthatmighthaveadheredtothesiliconlidduringtransportation.



Afterthisprocedure,theuppersealcanberemovedandthestepsoutlinedinthepurificationprotocolscanbeperformed.

Ifyoudonotdesiretouseall96wells,theplatesealscanbecutsothatonlythewellsthatareneededareexposed.Afterchromatogra-phy,theremovedportionoftheplatesealscanbereplacedandtheplatecanbestoredat4°Cuntiltheremainingwellsareused.Whenstored,theresininunusedwellsshouldbecoveredin20%ethanol.

2.HT96-WellPlateEquilibration a.Removethetopandbottomsealsfromtheplate. b.Placetheplateonthemanifoldandapplyvacuumtoremove

storagesolutionorcentrifuge5minat700xg. c.Add1mlofdeionizedwatertoeachwelloftheplate.Applyvacuum

orcentrifugetodrainthewaterfromthewells.Repeattwice. d.Add1mlof1XEquilibration/WashBuffertoeachwelloftheplate

andapplyvacuumorcentrifuge todrain thebuffer fromthewell.Repeattwice.

3.VacuumPurification Whenperformingvacuumpurification,adjustthevacuumtoobtain

aflowrateof1–2dropspersec(~100–200mmHgor2–4psi).Inaddition,avoidoverdryingtheresinwhichintroducesairbubblesandreducesperformance.

a.Apply1.5mlofthestartingsample(SeeSectionVII.E)perwell.Mixthesamplewiththeresinshortlybyvortexingtheplateorpipettingupanddowninsidethewells.Leavetheplateonicefor5–10minmixingsamplesevery2min.

b.Placetheplateonthevacuummanifold,applyvacuumandlettheexcessliquiddrainintothemanifold.Firmlypressallfoursidesoftheplatetotherubbergasketofthevacuummanifold.Ensurebyobservationthatallwellshavebeendrainedofbuffer.

c.RepeatSteps3.aand3.bifadditionalloadingisnecessary. d.Add1mlof1XEquilibration/WashBufferandsuspendtheresin

byvortexingtheplateorpipettingupanddowninsidethewells. e.Placetheplateonthevacuummanifold,applyvacuumandlet

theexcessliquiddrainintothemanifold.Firmlypressallfoursidesoftheplatetotherubbergasketofthevacuummanifold.Ensurebyobservationthatallwellshavebeendrainedofbuffer.

f.RepeatSteps3.dand3.etwice. g.Add1mlofHT96-WellPlateWashBuffer(SeeSectionIII)toeach

wellandsuspendtheresinbyvortexingtheplateorpipettingupanddowninsidethewells.




h.Placetheplateonthevacuummanifold,applyvacuumandlettheexcessliquiddrainintothemanifold.Firmlypressallfoursidesoftheplatetotherubbergasketofthevacuummanifold.Ensurebyobservationthatallwellshavebeendrainedofbuffer.

i.RepeatSteps3.gand3.hfivetimes. j.RemovetheHT96-WellPlatefromthevacuummanifold.Drain

thecollectedfiltratefromthevacuummanifold. k.PlaceaCollectionPlateinsidethevacuummanifoldandplace

theHT96-WellPlateonthevacuummanifold. Note:Beforeeluting,placetheplateoveraCollectionPlateoronthebaseseal.

l.Add200µlof1XElutionbuffer(SectionIII.A)andsuspendtheresinbyvortexingtheplateorpipettingupanddowninsidethewells.

m.Place the HT 96-Well Plate on the vacuum manifold, applyvacuum,andlettheeluatedrainintotheCollectionPlate.

n.Repeatelution(Steps3.mand3.n)twice. o.Determineamountofloadedandadsorbedproteinineachwell

byBradfordAssay(Bradford,1976).

4.CentrifugePurification Asavarietyofrotorsandcentrifugescanbeused,thefollowing

instructionsareonlygeneralguidelinesforsuccessfulpurification: • Donotutilizecentrifugalforcehigherthan700xg. • EnsureproperbalanceoftheHT96-WellPlate/CollectionPlate

insidetherotor. • Whenperformingthecentrifugeprocedurebelow,extraCollec-

tionPlatesarerecommended.SeeAdditionalMaterialsRequiredforinformationonobtainingcompatibleplates.

a.Add1.5mlofthestartingsampleperwell(SeeSectionVII.E).Mixthesamplewiththeresinbrieflybyvortexingtheplateorpipettingupanddowninsidethewells.Leavetheplateonicefor5–10minmixingsamplesevery2min.

b.Centrifugetheplatefor5min.Ensurebyobservationthatallwellshavebeendrainedofbuffer.

c.RepeatSteps4.aand4.bifadditionalloadingisnecessary. d.Add1mlof1XEquilibration/WashBufferandsuspendtheresin

byvortexingtheplateorpipettingupanddowninsidethewells. e.Centrifugetheplatefor5min.Ensurebyobservationthatall

wellshavebeendrainedofbuffer. f.RepeatSteps4.dand4.etwice. g.Add1mlofHT96-WellPlateWashbufferandsuspendtheresinby

vortexingtheplateorpipettingupanddowninsidethewells.




h.CentrifugetheHT96-WellPlatefor5min.Ensurebyobservationthatallwellshavebeendrainedofbuffer.

i.RepeatSteps4.gand4.hfivetimes. j.Drain thecollectedfiltrate fromtheCollectionPlateorusea

freshCollectionPlate(SeeSectionIII.E). k.PlacetheHT96-WellPlateontheCollectionPlateintherotor

andcentrifuge5min.Ensurebyobservationthatallwellshavebeendrainedofbuffer.

Note:Beforeeluting,ensurethattheplateisoveraCollectionPlateoronthebaseseal.

l.Add200µlof1XElutionbuffer(SectionIII.A)andsuspendtheresinbyvortexingtheplateorpipettingupanddowninsidethewells.

m.CentrifugetheHT96-WellPlateontheCollectionPlatefor5min.Ensurebyobservationthatallwellshavebeendrainedofbuffer.

n.Repeatelution(Steps4.land4.m)twice. o.Determineamountofloadedandadsorbedproteinineachwell

byBradfordAssay(Bradford,1976).

I. TALON® Magnetic Beads Purification ProtocolThisprotocolprovidesinstructionsforcarryingouttheTALONMagneticBeadspurificationinasingletube.ThebuffersusedinthisprocedureareeasilypreparedbydilutionfromthestockbuffersinourTALONMagneticBeadsBufferKit(Cat.No.635638).SeeSectionVforgeneralbufferpreparationguidelines.

1.Bufferpreparation a.Prepare5mlof1XEquilibration/WashBuffer • Ifthe5Xstockbufferisprecipitated,placebottleat37°C

for5min,thenshakeuntiltheprecipitatedissolves. • Dilute1mlof5Xstockwith4mlofH2O,confirmthatfinal

pHis7.0andcorrectpHifnecessary. b. Prepare0.5mlofElutionBuffer Add0.125mlof4XElutionBufferto0.375mlof1XEquilibra-

tion/WashbufferandconfirmthatthefinalpHis7.0.Anyunuseddilutedbuffercanbestoredandusedlater.

2.Generalconsiderationsforworkingwithmagneticbeads a.Useapipettetomixbufferthoroughlywiththebeads. b.Ifneeded,magneticbeadscanbemixedusingavortexer. c.Ifthereisagreatdealofliquid/bufferadheringtothesidesofthe

tube,centrifugethetubesinamicrofugebeforeplacingthemonamagneticseparator.

d.Ensurethatthebeadsareadheringtothesidesofthemagnetbeforeremovingthesupernatant.





3.Proteinpurificationundernativeordenaturingconditions a.Aliquot100–200µlofbeadsintoa1.5mlmicrofugetube. b. Placethetubeonamagneticseparatorfor1minandremove

storagebuffer. c. Add0.5mlofdeionizedwatertothebeads. d. Mixtheliquidandthebeadsthoroughlyusingapipette. e. Placethetubeonamagneticseparatorandremovethesuper-

natant. f. Toequilibratethebeads,add0.5mlof1XEquilibration/Wash

Buffer. g. Repeatstepsdande. h. Addthecelllysate(fromSectionsVII.ForVII.G)tothebeads. Note:Ifthecelllysatevolumeislessthan200µl,addsufficient1XEquilibra-

tion/WashBuffertobringthevolumeuptoatleast200µl.Thisisnecessarytoensurethoroughmixingofbeadswiththecelllysate,foroptimalbinding.

i. Mixonarotaryshakerfor30minatroomtemperature. Note:Iftheproteinisvulnerabletodegradationatroomtemperature,incubate

at4°Cfor1hr.ProteaseinhibitorsthatdonotcontainEDTAcanalsobeaddedduringtheincubation.

j. Placeonamagneticseparatorandcollectthesupernatant. k. Add0.5mlof1XEquilibration/WashBuffer. l. Mixthoroughlyandletitstandfor1minbeforeplacingona

magneticseparatorandcollectingthefirstwash. m. Repeatstepskandltwicetocollectthesecondandthirdwashes,

respectively. n. [Optional]: If necessary, repeat steps k and l under more

stringent conditions using 0.5 ml of 5–10 mM imidazole in1XEquilibration/WashBuffer(sectionV.D)

o. Toelutetheprotein,add50µlofElutionBuffer.ThevolumeofElutionBuffercanbevarieddependingontheamountofbeads used. 50 µl of elution buffer can be used for elutingfrom200µlofbeadsuspension.Mostoftheproteinwilleluteinthisfraction.Smallervolumes,suchas25µlcanbeusedifaconcentratedsampleisneeded.Volumesbelow25µlmaybedifficulttohandle.

p. Mixfor5minandcollectEluate1. q. Addanother50µlofElutionBuffer r. Mixfor1minandcollectEluate2.




s.Ifnecessary,stepsqandrcanberepeatedtwicetoensurethatproteinrecoveryismaximized.Inaspecificinstance,whenusing200µlofbeadsuspension,60%ofthetotalproteinwaselutedinthefirst50µlfraction,20%inthesecond,10%inthethird&5%inthefourth.

t. UsespectrophotometricandSDS-PAGEanalysestodeterminewhichfractionscontainthebulkofthepolyhistidine-taggedprotein.

Note:ABradfordproteinassayisrecommendedformeasuringproteinyields.Since thedetergents in thexTractorBuffermay interferewith theBradfordassay,itisadvisabletoruntheoriginallysateandnon-adsorbedfractionata1:5dilutionoruseaBCAassayforundilutedsamples.



IX. Resin Washing, Reuse, and Storage

Generally,reuseTALON®Resins3–4timesbeforediscardingorcompleteregeneration.Theexactnumberofusesvariesamongpreparationsandapplicationbecauseofdifferencesinredoxpotential,organiccomplexity,anddebriscontent.Toavoidpossiblecross-contamination,useaparticularaliquotofresintopurifyasingletypeofpolyhistidine-taggedprotein.

Important precautions• TALONspin™Columnsarenotreusable.• DonotstoreTALONResinindenaturantssuchas6Mguanidinium.• DonotstoreTALONResinwithbound imidazole: the resinshouldbe

washedwithMESBuffer(pH5.0)describedinSectionIII,whichisrequiredbeforereusetoremovetheboundimidazole.

A. Stringent Wash (optional) 1.Washresinwithfourbedvolumesof6Mguanidinium(pH5.0)+

1%nonionicdetergent. 2.RinseresinwithfivebedvolumesofdistilledH2O. 3.Storeresinat4°Cin20%nonbufferedethanolcontaining0.1%azide.

B. Removing Imidazole 1.Washresinwithfivebedvolumesof20mMMESBuffer(pH5.0)

containing0.1MNaCl. 2.RinseresinwithfivebedvolumesofdistilledH2O. 3.Storeresinat4°Cin20%nonbufferedethanolcontaining0.1%azide.

C. Regeneration of Superflow Columns Purificationofpolyhistidine-taggedproteinsusingimidazolegradients

willcausethecolumntotakeonapurplishhue.Washingthecolumnwith5–10columnvolumesof20mMMESBuffer(pH5.0)willrestorethenormalpinkcolorandbringtheabsorbanceat280nmback to theoriginalbaselinelevel.AfterequilibratingthecolumnwithEquilibra-tion/WashBuffer,thecolumnisreadyforreuse.

D. Complete Regeneration 1.Striptheresinofcobaltionsbywashingwith10bedvolumesof

0.2MEDTA(pH7.0). 2.WashexcessEDTAfromtheresinwithanadditional10bedvolumes

ofdoubledistilledH2O(ddH2O). 3.Chargetheresinwith50mMCoCl2solution(10bedvolumes). 4.Washresinwith7bedvolumesofddH2Ofollowedby3bedvolumes

of300mMNaCl,and3bedvolumesofddH2Otoremoveexcesscobaltmetalions.



5.EquilibratetheresinwithEquilibration/Washbuffer(10bedvolumes). 6.Ifyouplanttouseβ-mercaptoethanolinsubsequentbuffers/pro-

cedures,thenre-equilibratetheresinasfollowsbeforeproceedingwithfutherpurifications:

a.WashtheresinwithatleasttwobedvolumesofEquilibration/WashBuffer.

b.Re-equilibratetheresinwithEquilibration/WashBuffercontainingβ-mercaptoethanol.

E. TALON Magnetic Beads TALON Magnetic Beads are for single use only.They cannot be

regenerated.

IX. Resin Washing, Reuse, and Storage continued



X. Troubleshooting Guide

A. Protein Expression

1.Noexpression

• Bad vector construct Checksequenceofthevector.

• Bad transformation Makeaplasmidminiprepandconfirmsequence.

• No inducing agent added to culture to induce expression

2.Apparentlowexpression

• Insoluble overexpressed protein Usedenaturingextractionandpurificationconditionsorreduce

expressionlevelsbyloweringtheamountofinducer.

• Unsuitable expression conditions Checkcellgrowthandinducerconcentration;checkforwild-type

(nontransformed)orantibioticresistantcells.

• Protein is secreted UsefermentationliquidasstartingsampleforIMACafterproper

buffering.

B. Loading/Washing

1.Polyhistidine-taggedproteinelutesinthewashbuffer

• Problems with vector construction Ensurethatproteinandtagareinframe.

• Buffer is not optimal CheckthepHandcompositionofallbuffers.Usealowerstringency

washbufferforallwashingsteps.Forexample,slightlyincreasethepHofthewashbufferorloweritsimidazoleconcentration.

• Protein degraded during extraction a.Usemildextractionconditions in thepresenceofprotease

inhibitors (e.g.,β-MEandEDTA)at4°C. Be sure to remove EDTA before applying to TALON®Resin.

b.MakeC-terminalconstruct.

c.Workquicklyat4°Ctoreducethetimeforinitialpurificationsteps.

• Reagent interferes with binding SeeAppendixAforreagentcompatibilities.Diluteanaliquotof

lysate(1:10),orsonicate,andcheckbindingonasmallscale.Tryusingadifferentpolyhistidine-taggedproteinasacontrol.

• Tag is not accessible under native conditions Iftheproteinfailstobindundernativeconditions,treatasmall



aliquot(<1ml)with6Mguanidiniumandbindto50µlofresin.Thenfollowthemini-scaleprocedureinAppendixB.Ifthetargetproteinbindstotheresinunderthedenaturingconditions,thentrytomovethetagtotheotherterminusoftheproteinwhereitmaybemoreexposed.

2.Highbackpressureduringloadofsample

• High viscosity due to presence of DNA UseDNaseIordilutesamplefivefold(SectionVIII.A.8).

C. Elution

1.Highamountofco-elutedimpurities

• Insufficient wash UselargervolumesofEquilibration/WashBuffer.

• Buffer compositions are not optimal a.Checkbuffersusedforsamplepreparationandwashsteps. b.Check pH.The Equilibration/Wash Buffer should be pH 7.0.

Contaminantswillco-eluteinbuffers<pH7.0. c. Increasevolumeofwashbufferandcontinuetowashresin

beduntiltheA280dropstozero. d.Increasecounterionconcentrationupto0.5MNaClorKClto

inhibitnonspecificionicinteractions. e.Addethyleneglycolorglyceroltoinhibitnonspecifichydro-

phobicinteractions. f. Addsmallamountsofnonionicdetergent(s);thisisparticularly

importantwhenisolatingproteinsfromaeukaryoticexpres-sionsystem.

g.Add5–10mMimidazoletotheEquilibration/WashBufferanduseitasanintermediatewashstepbeforeelution.

• Proteolytic product Usemildextractionconditionsinpresenceofproteaseinhibitors

(e.g.,β-MEandEDTA)at4°C. Remove EDTA before applying to TALON®Resin.Proteinscanbeextractedinpresenceofproteaseinhibitorsspeciallydesignedforpurificationofhistidine-taggedproteinsastheydonotcontainEDTA.

• Covalent attachment (Cys-Cys) of impurities to the protein Use5–10mMofβ-MEintheEquilibration/WashofBuffer.

• Co-purifying histidine rich proteins a.ForHATproteins,useenterokinasetoremoveHATtagand

rerunIMACwithmixture.Targetproteinwillpassthroughthecolumn,whileimpuritiesandtagwillbeadsorbed.

X. Troubleshooting Guide continued

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT1320-1 �0 Version No. PR6Z2142


Note:Removechelatingligandsbygelfiltrationbeforeloadingtheproteolytic mixtureontoTALONResin.

b.Usesecondpurificationprinciple,suchassizeexclusion,ionexchange,hydrophobic,orthiophilicchromatography.

• Protein sample is too concentrated and/or viscous Dilutesample1:5or1:10withadditionalbufferandcentrifuge

againbeforeproceeding.Also,seethenoteonreducingsampleviscosityaftersonicationinSectionVII.A.8.

2.ExcessivebackgroundafterTALONspin™Columnprocedure

• Sample is too viscous a.Reducetheviscosityofthesample(SectionVIII.A.8). b.DiluteclarifiedsamplewithanequalvolumeofEquilibration/

WashBufferandloadastwoaliquots. c. Increasenumberof1mlwashes. d.UseEquilibration/WashBuffer(pH7.0). e.Add1–5mMimidazoletoEquilibrationBuffer,pH8.0anduse

itasanintermediatewashstepbeforeelution. f. Tore-purifyasample,performthefollowingafterStep37in

SectionVIII.G: 1.Add4volumesofEquilibration/WashBuffertosemi-purifiedsample. 2.LoadsampleontoanotherTALONspin™Column. 3.Washtwicewith1mlofEquilibration/WashBuffer.

4.Eluteasbefore(StepsVIII.G.30–35).

3.Columnceasestoflow

• Filter is clogged with subcellular debris Changecolumnfiltersandcentrifugesampleat12,000xgfor

20–30minat4°C.

• Proteins precipitated on the column Use a mild detergent such as Decanoyl-N-methylglucamide

(MEGA-10, Sigma Cat. No. D6277) in the Equilibration/WashBuffer.

• The lower resin bed support may be clogged with cellular debris a.Removeresinfromcloggedcolumnandresuspend.Thenwash

itinabatchformatandtransfertoafreshcolumn. b.Useasyringefilledwithwashbufferorreversethepumpon

thecolumntogentlyrunthecolumnbackwards.Inaddition,test for tubingblockages inasimilarmanner.Applygentlepressure.Donotexceeda1drop/secflowrate.


Protocol No. PT1320-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR6Z2142 �1


4.Polyhistidine-taggedproteinsdonotelute

• Elution Buffer is less than optimal

a.Elutewith150mMimidazoleorpH4.0buffer.

b.Forreallytoughelutionproblems,youcanstripofftheproteinusing100mMEDTA(pH8.0);however,doingsowillremovethecobaltfromtheresinanddeposititinyourproteinsample.

c. Add1–5mMβ-MEtoreducedisulfidelinkages.Supplementbufferwith1%nonionicdetergent.

d.Purifypolyhistidine-taggedproteinunderdenaturingconditions.

D. Changes in Resin

1.Resinchangesfrompinktowhite—LossofCo2+

• Presence of chelators in sample Removechelatorsfromsamplebygelfiltration RegenerateresinasdescribedinSectionIX.D.

2.Grayorbrownresin

• TALON® Resin exposed to reducing agents or high concentration of β-ME

Completelyremovereducingagents,suchasDTEorDTT,orifpossible,bygelfiltrationwithβ-ME.Reduceβ-MEconcentration(≤5mM).

3.Resinparticlesaggregateorexhibitchangeinconsistency

• DNA cross-linking a.Increaseionicstrengthofthebuffersbyusing

≤500mMNaClorKCl.

b.VigorouslysonicatesampletoshearDNA.

c. Pretreatsamplewith100µg/mlDNaseIat30°Cfor30min.

d.Dilutesample1:5–1:10withbuffer,andrepeat.

e.Avoidlong-termstorageindenaturants.

E. Analysis

1.Highbackgroundonsilver-stainedgels

• Nucleic acid a.Supplementbufferwith0.2–0.5MNaClorKCl.

Repeatpurification.

b.ShearDNAmorevigorously.

c. UseDNaseIintheextractionprocedure.





2.Nonfunctionalprotein

• Protein was damaged by sonication a.Conduct a time-course assay to determine the minimum

sonicationtimeneededtodisruptthecellswhilemaintainingthe native protein/enzyme function. For example, sonicatesamplesatamedium-highsettingfor0,20,and30sec.ThenperformproteinorenzymefunctionassaysandmeasuretheA280ofeachsample.

b.Performthelysisorsonicationprocedureonice.

F. Reuse

1.Bindingdropsbeloworiginalcapacity • Lysate contains naturally occurring reducing agent or a nonspe-

cific polyanion may be obscuring the metal binding sites. a.Usealargervolumeofpreviouslyusedresin. b.Replaceusedresinwithfreshresin. c. Washresinwith6Mguanidinium(pH5.0)+1%TritonX-100

orSDS,andre-equilibratebeforeuse.

G. Application of samples prepared from overnight cultures to TALON Magnetic Beads.

1.Noproteinbindstothebeadswhenusingovernightculture.CheckthepHandensurethatitisbetween7–8.

2.Thebeadsfailtomigratetothemagnet,duetothehighviscosityofthesolution.

a. AddsufficientDNase(1unit/mlofculture)andmixthoroughlybeforeaddingbeads.

b. Dilutethesamplefurtherwith5XEquilibration/WashBuffertoobtainafinalconcentrationof1XEquilibration/WashBuffer.

Protocol No. PT1320-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR6Z2142 �3


Bradford,M.M.(1976)Arapidandsensitivemethodforthequantificationofmicrogramquanti-tiesofproteinutilizingtheprincipleofprotein-dyebinding.Anal. Biochem.72:248–254.

Bush,G.L.,Tassin,A.-M.,Friden,H.&Meyer,D.I.(1991)Secretioninyeast:purificationandin vitrotranslocationofchemicalamountsofprepro-alpha-factor. J. Biol. Chem.266:13811–13814.

Chaga,G.,Bochkariov,D.E.,Jokhadze,G.G.,Hopp,J.&Nelson,P.(1999)Naturalpoly-histi-dineaffinitytagforpurificationofrecombinantproteinsoncobalt(II)-carboxymethylaspartatecrosslinkedagarose.J. Chromatogr. 864:247–256.

Hemdan,E.S.&Porath,J.(1985a)DevelopmentofImmobilizedMetalAffinityChromatographyII.Interactionofaminoacidswithimmobilizednickeliminodiacetate.J. Chromatogr. 323:255–264.

Hemdan,E.S.&Porath,J.(1985b)DevelopmentofImmobilizedMetalAffinityChromatographyIII.Interactionofoligopeptideswithimmobilizednickeliminodiacetate.J. Chromatogr.323:265–272.

Hochuli,E.,Döbeli,H.&Schacher,A.(1987)Newmetalchelateadsorbentselectiveforproteinsandpeptidescontainingneighboringhistidineresidues.J. Chromatogr.411:177–184.

Hochuli, E., Bannwarth,W., Döbeli, H., Gentz, R. & Stüber, D. (1988) Genetic approach tofacilitatepurificationofnovel recombinantproteinswithanovelmetalchelateadsorbent.Bio/Technology 6:1321–1325.

Kasher,M.S.,Wakulchik,M.,Cook,J.A.&Smith,M.(1993)One-steppurificationofrecom-binanthumanpapillomavirusType16E7oncoproteinanditsbindingtotheretinoblastomageneproduct.BioTechniques14:630–641.

Porath, J. (1985) Immobilized Metal IonAffinity Chromatography—A Powerful Method forProteinPurification.InModern Methods in Protein Chemistry,(pp.85–95).H.Tschelsche(Ed.),(Berlin&NY:WalterdeGruyter&Co).

Porath,J.,Carlsson,J.,Olsson,I.&Belfrage,G.(1975)Metalchelateaffinitychromatography,anewapproachtoproteinfractionation.Nature258:598–599.

Sambrook,J.&Russell,D.W.(2001)Molecular Cloning: A Laboratory Manual (ColdSpringHarborLaboratory,ColdSpringHarbor,NY).

Sulkowski,E.(1985)PurificationofproteinsbyIMAC.Trends Biotechnol.3:1–7.

TALON®MagneticBeads(October2005)ClontechniquesXX(2):14.

Zhao,Y.-J.,Sulkowski,E.&Porath,J.(1991)Surfacetopographyofhistidineresiduesinlyso-zymes.Eur. J. Biochem.202:1115–1119.

XI. References



Appendix A. Reagent Compatibilities and Incompatibilities

A. Compatible reagents TableIIIshowsthemaximumconcentrationsofeachreagenttestedat

Clontech.Higherlevelsmaybeacceptable,buttheyshouldbetestedbeforeuse.Notethatsomeofthesereagentsmaypartiallyorcom-pletelydenatureyourprotein.

table iii. reagent compatibility

Reagent Acceptable Concentration

β-Mercaptoethanola 10mM(withcaution) CHAPSb 1%(withcaution) Ethanolc 30% Ethyleneglycol 30% HEPES 50mM Glycerol 20% Guanidiniuma 6M imidazoled 200mMatpH7.0–8.0,forelution KCl 500mM MES 20mM MOPS 50mM NaCl 1.0M NP-40 1% SDSb 1%withcaution TRISe 50mM Triton-X100 <1% Urea 8Ma Useresinimmediatelyafterequilibratingwithbufferscontainingthesereagents.Otherwise,

theresinwillchangecolor.Donotstoreresininbufferscontainingthesereagents.b IonicdetergentslikeCHAPS(3-[(30Cholamidopropyl)-dimethylammonio]-1-propane-sulfo-

nate),SDS(sodiumdodecylsulfate),andsarkosylarecompatibleupto1%.However,duetotheirchargednature,youshouldanticipateinterferencewithbinding.

c Ethanolmayprecipitateproteins,causinglowyieldsandcolumnclogging.d Imidazolecannotbeusedatconcentrationshigherthan5–10mMforloadingpolyhistidine-

taggedproteins,becauseitcompeteswiththehistidinesidechains(imidazolegroups)forbindingtotheimmobilizedmetalions.

e TRIScoordinatesweaklywithmetalions,causingadecreaseincapacity.

Protocol No. PT1320-1 www.clontech.com Clontech Laboratories, Inc. Version No. PR6Z2142 ��


B. Incompatible reagents Thesereagentsareincompatibleatanyconcentration:

• DTT(dithiothreitol)andDTE(dithioerythritol) Note:Useofstrongreducingagentswillinterferewiththebindingofthecobalt

metalionstotheresin.

• EDTA(ethylenediaminetetraaceticacid)andEGTA(ethyleneglycol-bis([β-amino-ethylether])

Note:AlthoughyoucanuseEDTAatindicatedpoints,itmustberemovedfromthesamplebygelfiltrationpriortoapplyingittoTALON®Resins.

Appendix A. Reagent Compatibilities and Incompatibilities



Appendix B. Mini-Scale Protein Purification Protocol

Mini-scaleproteinpurificationisidealforanyofthefollowing: (a)checkingforapolyhistidine-taggedprotein (b)determiningexpressionlevels (c)testingbufferconditionsYoucanuseaTALON®SingleStep(Cat.No.635628or635631)forproteinminiprep,oryoucanuseaTALONspin™Column(Cat.No.635601)withthisprocedure.Werecommendthatyousetasideasampleaftereachcriticalstepoftheprocedure,andanalyzeallsamplesbySDS/PAGE.Important• Thisprotocolisnotintendedforobtaininghighlypurifiedpolyhistidine-

taggedproteinsamples.Furthermore,proteinsampleselutedwithEDTA(Step19,below)willcontaincobaltandEDTA,whichmayseriouslyinhibitenzymeactivityandmaycausetheproteintoprecipitate.

• ThisprotocolwasoptimizedusingdenaturingconditionsatpH8.0.Ifyouwishtoobtainnativesamples,thensubstitutebuffersaccordingly.Youmayalsoneedtouselysozyme(0.75mg/mlofnativebuffer)tocompletelydisruptthecellsinStep5.

1.Transfer1mlofexpressionculturetoa1.5mlmicrocentrifugetube. 2.Centrifugeat14,000rpmfor2min. 3.Removeanddiscardsupernatant. 4.Add0.5mlofDenaturingEquilibrationBuffer(pH8.0). 5.Vortexuntilcellpelletiscompletelydissolved. 6.Centrifugeat14,000rpmfor5mintopelletanyinsolubledebris. 7.Setaside50µlofthesupernatantforlateranalysis.Transferthe

remainderofthesupernatanttoaclean1.5mltubecontaining50µlofprewashedTALON®Resin,preparedasdescribedinSectionVIII.B.Steps1–7.Startwith100µlofresuspendedslurry.

8.Agitatesampleatroomtemperaturefor10min. 9.Centrifugeat14,000rpmfor1mintopelletprotein/resincomplexes. 10.Carefully remove the supernatant and set aside 50 µl for later

analysis.Ahighproteinconcentrationinthissampleindicatesaproblemwithproteinbinding.

11.Add1mlofDenaturingEquilibrationBuffer. 12.Vortexforafewsec. 13.Centrifugeat14,000rpmfor1mintopelletresin. 14.Removethesupernatantandsetaside50µl(“firstwash”)forlater

analysis.Discardtheremainderofthesupernatant.



Appendix B. Mini-Scale Protein Purification...continued

15.RepeatSteps11–14.Setaside50µlforanalysis. 16.Eluteboundpolyhistidine-taggedproteinbyadding50µlofElution

Buffertotheresin/proteinpelletandbrieflyvortexing. 17.Centrifugebrieflyat14,000rpm. 18.Carefully remove the supernatant containing the polyhistidine-

taggedprotein. 19.RepeattheSteps16–18.Alternatively,ifyouonlyintendtodetermine

theconcentrationofpolyhistidine-taggedproteininyoursample,youcanachieveamorecompleteelution,andthus,amoreac-curateproteinquantificationbyelutingwithEDTAasfollows:

a. Add50µlof100mMEDTA(pH8.0)andvortexbriefly. b. Centrifugebrieflyat14,000rpm. c. Carefullyremovethesupernatantcontainingthe6xhistidine-

taggedprotein. Note:EDTAremovesboundmetalfromtheresin:theproteinsamplewillcontain

cobalt,andtheTALON®Resincannotbereused.

20.Add12µlof5XSDS/PAGEsamplebuffertoeachofthesavedsamples. Note:Thesamplebufferwillreducemultimerstomonomers;thus,onlyasingleband

willbevisibleonanSDS/PAGEgel,evenfornaturallyhomologousmultimericproteins.

21.Heatsamplesat95–98°Cfor5min. 22.LoadsamplesandanalyzeonanSDS/PAGEgel.

Clontech Laboratories, Inc. www.clontech.com Protocol No. PT1320-1 �� Version No. PR6Z2142


Figure 6. pHAT10/11/12 combined vector map and MCS. Uniquerestrictionsitesareinbold.ThesequenceofpHAT10isshown.TheasteriskindicatestheinsertionpointofadditionalbasesinpHAT11(G)andpHAT12(GG)thatalterthereadingframeoftheMCS.ThesevectorsencodeanovelpolyhistidineepitopetagthatenablespurificationofexpressedproteinsatneutralpH.ThepHATVectorsallowproteinpurificationunderbothnativeanddenaturingconditions.TheHATepitopeisanaturallyoccurring,19-amino-acidsequencefromthechickenlactatedehydrogenaseprotein.Thissequenceofnonadjacenthistidineresidueshas loweroverallchargethantagswithconsecutivehistidineresidues,suchasthe6xhistidinetag.Asaresult,HAT-proteinfusionsexhibitsolubilitythatmorecloselyresembleswild-typeproteinswhilestillpossessingstrongaffinityforimmobilizedmetalions.TheuniquebindingcharacteristicsoftheHATsequenceallowbothimidazole-andpH-gradientpurificationofproteinsundernativeconditionsatneutralpH(7.0),aswellasunderdenaturingconditions.TheHATsequenceandanenterokinase(EK)cleavagesitehavebeenincorporatedintothepUC19backbone.TheEKsiteallowsforoptionalremovaloftheHATsequencefromthepurifiedproteinbytreatmentwithenterokinase.RestrictionsitesallowexcisionoftheHATsequence,withorwithouttheEKsite,forcloninginothervectors.

Appendix C: Vector Information

pHAT10/11/122.� kb

MCS

Ampr

Plac

pUCori

HAT

EKsite

A AGC TTG AAG GAT CAT CTC ATC CAC AAT GTC CAC AAA GAG GAG CAC GCT CAT GCC CAC AAC AAG

ATCGATGACGATGACAAAGTCGACGGATCCCCGGGTACCGAGCTCGTAATTAGCTGAATTCSal I Sma I Sac IKpn IBamH I EcoR I

Hind III

Cla I

Ser Leu Lys Asp His Leu Ile His Asn Val His Lys Glu LysAsnHisHis Ala HisAlaGlu

EK cleavage site

HAT

*



Appendix C: Vector Information continued

Figure 7. pHAT20 combined vector map and MCS. Uniquerestrictionsitesare inbold.ThesequenceofpHAT20isshown.ThisvectorencodesanovelHistidineAffinityTag(HAT)thatenablespurificationofexpressedproteinsatneutralpH.ThepHATVectorsallowproteinpuri-ficationunderbothnativeanddenaturingconditions.TheHATepitopeisanaturallyoccurring,19-amino-acidsequencefromthechickenlactatedehydrogenaseprotein.Thissequenceofnonadjacenthistidineresidueshasloweroverallchargethantagswithconsecutivehistidineresidues,suchasthe6xhistidinetag.Asaresult,HAT-proteinfusionsexhibitsolubilitythatmorecloselyresembleswild-typeproteinswhilestillpossessingstrongaffinityforimmobilizedmetalions.TheuniquebindingcharacteristicsoftheHATsequenceallowbothimidazole-andpH-gradientpurificationofproteinsundernativeconditionsatneutralpH(7.0),aswellasunderdenaturingconditions.TheHATsequenceandanenterokinase(EK)cleavagesitehavebeenincorporatedintothepUC19backbone.TheEKsiteallowsforoptionalremovaloftheHATsequencefromthepurifiedproteinbytreatmentwithenterokinase.RestrictionsitesallowexcisionoftheHATsequence,withorwithouttheEKsite,forcloninginothervectors.

pHAT202.� kb

Ampr

MCS (221–256)

pUC ori

Plac

ApaL I(481)

EK site

Hind III (141)

ApaL I(978)

ApaL I(2224)

EcoR I (262)

HAT

A AGC TTG AAG GAT CAT CTC ATC CAC AAT GTC CAC AAA GAG GAG CAC GCT CAT GCC CAC AAC AAG

ATC GAT GAC GAT GAC AAA

Hind III

Ser Leu Lys Asp His Leu Ile His Asn Val His Lys Glu LysAsnHisHis Ala HisAlaGlu

HAT

Cla I

EK cleavage siteGTT AAC CGG TCC CCG GGT ACC GGG CCC GGC CGG CCHpa I Age I Sma I Kpn I

Eag I Fse INae I

150•

160•

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TALON Metal Affinity Resins User Manual - … · 2017-09-12 · Using theTALON® Metal Affinity...

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Transcript of TALON Metal Affinity Resins User Manual - … · 2017-09-12 · Using theTALON® Metal Affinity...