protino® glutathione agarose 4b

MACHEREY-NAGEL

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Purification of Gst-tag proteins

User manualProtino® Glutathione Agarose 4B

May 2013 / Rev. 04

A035

480

/ 094

0.3

3

Purification of GST-tagged proteins

MACHEREY-NAGEL – 05 / 2013, Rev. 04

Table of contents

1 Components 41.1 Kit contents and storage 41.2 Additional material to be supplied by user 4

2 Product description 52.2 Culture size 62.3 Preparation of buffers 8

4 Protocols 104.1 Preparation of cleared E. coli lysates 104.2 BatchpurificationofGST-taggedproteins 124.3 Batch/gravity-flowpurificationofGST-taggedproteins 144.4 SpincolumnpurificationofGST-taggedproteins 154.5 FPLC™purificationofGST-taggedproteins 17

5 Regeneration and storage 19

6 Appendix 206.1 Troubleshooting 206.2 Convertingfromlineartovolumetricflowratesandviceversa 226.3 Ordering information 236.4 Productuserestriction/warranty 24



1 Components

1.1 Kit contents and storage

Protino® Glutathione Agarose 4B

REF 745500.10 745500.100

Protino®GlutathioneAgarose4BVolume of settled beaded agarose (bed volume)

10 mL 100 mL

User manual 1 1

Protino® GlutathioneAgarose 4B is supplied as a 75% (v/v) aqueous suspensioncontaining20%ethanoltoinhibitbacterialgrowth.

Shipping and storage of Protino® Glutathione Agarose 4B:

Theproductisshippedatambienttemperature.Upon receipt Protino®GlutathioneAgarose4Bshouldbestored at 4 °C and is stable up to 3 years. Do not freeze.

1.2 Additional material to be supplied by user

• PBS(10mMNa2HPO4,1.8mMKH2PO4,2.7mMKCl,140mMNaCl,pH7.3)

• ElutionBuffer(50mMTris-HCl,10mMglutathione,pH8)

• Lysozyme(requiredforcellextractpreparation,seesection4.1)

• Appropriate centrifuge tubes, collecting tubes

• Appropriate centrifuge

• Spincolumnsorchromatographycolumns

5



2 Product descriptionProtino®GlutathioneAgarose4Bisanaffinitychromatographymediumwhichenablesfastsinglesteppurificationofglutathione-S-transferase(GST)fusionproteinsandotherglutathione-bindingproteins.PurificationoffusionproteinsfromawholecelllysateisbasedonthestrongaffinityoftheGSTmoietyforglutathione,whichisimmobilizedonagarosebeads.GST-taggedproteinsareelutedundermild,non-denaturing,conditionsusingneutral-pHbufferscontainingfreeglutathione.Thepurificationprocesspreservesprotein antigenicity and functionality. If removal of theGST-tag is necessary, fusionproteinsmaybecleavedusingasite-specificprotease.

Protino®GlutathioneAgarose4Bcanbeusedinbatchbindingstudiesand / or packed bedchromatographyexperiments.PackedcolumnsmaybeoperatedbygravityfloworanyliquidchromatographysystemsuchasFPLC™system.

2.1 Specifications

Table 1: Specifications Protino® Glutathione Agarose 4B

Application Batch binding Gravityflowcolumnchromatography MPLC / FPLC™

Form 75%(v/v)aqueoussuspensioncontaining20 %ethanol

1 mL of settled agarose beads (1 mL bed volume) correspondsto1.333mLoforiginal75 %suspension

Matrix 4 %beadedagarose

Ligand Glutathione,linkedviasulfuratom

Spacer arm 12 atoms

Bead size 90 μm

Binding capacity1 >8mgrecombinantGST/mLsettledagarose

Recommended flow rates

1mLbedvolume(columnwith6.6mminnerdiameter) Sampleloading2: 0.2–1.0 mL/min Washing and elution: 1.0 mL/min

10mLbedvolume(columnwith16mminnerdiameter) Sampleloading2: 0.5–5.0 mL/min Washing and elution: 5 mL/min

1 BindingcapacitywillvaryforeachGST-taggedprotein.2 SlowflowratesarerecommendedfortheloadingsteptoallowmaximalbindingoftheGST-taggedprotein.



Table 1: Specifications Protino® Glutathione Agarose 4B

Maximum linear low rate3

250 cm/h

Chemical stability Protino®GlutathioneAgarose4Bwithstandsincubationin0.1MacetatepH4,0.1MNaOH,70 %ethanol,or6Mguanidinehydrochloridefor2hoursatroomtemperaturewithoutsignificantlossofproteinyield

Storage temperature 4–8 °C

Storage solution 20 %ethanol

Note Do not autoclave the gel.

2.2 Culture size

The yield of GST-tagged proteins depends on various parameters, such as natureof the fusion protein, expression host, culture conditions, etc. However, somerecommendations on protein load and culture size can be given (see Figure 1).Culturevolumerequirementsarebasedonthefollowingassumptions:

• Protino®GlutathioneAgarose4Bhasabindingcapacityof8mgofrecombinantGSTpermLofsettledagarose.

• Typically,theexpressionlevelofGST-taggedproteinsishighrangingfrom10to 50 mg/L of E. coli culture.

• Asastartingpointwerecommendtousethecell lysatefroma160–800mLE. coli culture per 1 mL of settled Protino®GlutathioneAgarose4B.

• SinceProtino®GlutathioneAgarose4Bissuppliedas75%(v/v)suspension,1 mL of settled agarose beads (1 mL bed volume) corresponds to 1.333 mL of original75%suspension.

3 Forconvertingfromlineartovolumetricflowrateseesection6.2.

7



1 mL Protino®GlutathioneAgarose4B1 (1 mL bed volume)

Load ~ 8 mg protein

Expression level 10mg / L

Expression level 50mg / L

Use 800 mL culture (~ 3.2 g cell pellet2)

Use 160 mL culture (~ 0.64 g cell pellet2)

Resuspend in upto16mLPBS3

Resuspend in upto3.2mLPBS3

~ 20 mL protein lysate ~ 4 mL protein lysate

Figure 1: Required culture volumes for 1 mL settled Protino® Glutathione Agarose 4B1

1 1mLbedvolumecorrespondsto1.333mLof75%(v/v)Protino®GlutathioneAgarose4Bsuspension.2Onaverage,250mLofculturewillproduceapproximately1gofpelleted,wetcells.31gcellsmaybelysedin2–5mLPBS,seesection4.1.



2.3 Preparation of buffers

Prepare the following working solutions.

For the preparation of cell lysates, 5 mL of PBS per g of E. colipelletwetmassarerequired.

Batch, gravity-flow, spin column applications:Per 1 mL of settled Protino®GlutathioneAgarose4B,approximately50 mL of PBS and 5 mL of Elution Bufferarerequiredforthepurificationprocedure.

FPLC™ applications (1 mL bed volume):10mLofPBSarerequiredtoequilibrate thecolumn,10mLofPBSarerequiredtowashthecolumnaftersampleapplication.10mLofElutionBufferarerequiredfortheelution step.

NotethatadditionalvolumesofPBSandElutionBuffermustbepreparedtoflushlinesand pumps depending on the chromatographic system: E.g., prepare approximately 250 mL of PBS and 150 mL of Elution Buffer.

Usehigh-puritychemicalsandwaterforpreparingthebuffers.Forbestresults,filterbuffers through a 0.45 μmfilterbeforeuse.

PBS (1 liter):

10mM Na2HPO4 1.780g Na2HPO4 • 2 H2O Mr = 177.99g/mol1.8mM KH2PO4 0.245 g KH2PO4 Mr = 136.09 g/mol2.7mM KCl 0.201 g KCl Mr = 74.55g/mol140mM NaCl 8.182 g NaCl Mr = 58.44 g/mol

AdjustpHto7.3

Elution Buffer (1 liter):

50mM Trisbase 6.06 g Trisbase Mr = 121.14g /mol10mM glutathione 3.07g glutathione Mr = 307.3g /mol

Adjust pH to 8.0 Preparefreshdailyandstoreat4 °C

9



3 Safety instructionsOnlyharmfulfeaturesdonotneedtobelabeledwithHandPphrasesupto125mLor 125 g. Mindergefährliche Eigenschaften müssen bis 125 mL oder 125 g nicht mit H- und P-Sätzen gekennzeichnet werden.

Component Hazard contents GHS symbol Hazard phrases

Precaution phrases

Inhalt Gefahrstoff GHS Symbol H-Sätze P-Sätze

GlutathioneAgarose 4B

Ethanol5–20% Warning 226 210, 233, 403+235Ethanol 5–20 % Achtung

Hazard phrasesH 226 Flammableliquidandvapour.

Flüssigkeit und Dampf entzündbar.

Precaution phrasesP 210 Keepawayfromheat/sparks/openflames/hotsurfaces.–Nosmoking.

Von Hitze/Funken/offener Flamme/heißen Oberflächen fernhalten.P 233 Keep congtainer tightly closed.

Behälter dicht verschlossen halten.P 403+235 Storeinawell-ventilatedplacxe.Keepcool.

Kühl an einem gut belüfteten Ort aufbewahren.

ForfurtherinformationpleaseseeMaterialSafetyDataSheets (www.mn-net.com). Weiterführende Informationen finden Sie in den Sicherheitsdatenblättern (www.mn-net.com).

Thesymbolshownonlabelsreferstotheprecautionphrasesofthissection.Das auf Etiketten dargestellte Symbol weist auf die P-Sätzen dieses Kapitels hin.


Protino®GlutathioneAgarose4B

4 Protocols

4.1 Preparation of cleared E. coli lysates

1 Cultivate and harvest cells

• Asastartingpointwerecommendtoprepare160–800mLE. coli expression cultureforthepurificationof8mgofGST-taggedproteinusing1mLbedofsettled Protino®GlutathioneAgarose4B(seesection2.2).

• Harvest cells from an E. coli expression culture by centrifugation at 4,500–6,000 x gfor15minat4 °C.Removesupernatant.

• Cellpelletsmaybestoredat-20 °Cor-80 °Cuntilneeded.

2 Resuspend bacteria cells

• ThawthecellpelletfromanE. coli expression culture on ice (if frozen).• Resuspend1gofpelleted,wetcellsin2–5mLPBS.Pipetteupanddown,

orstiruntilcompleteresuspensionwithoutvisiblecellaggregates.Performthis step on ice.

3 Lyse cells

• Addlysozymetoafinalconcentrationof1mg/mL.• Stirthesolutiononicefor30min.• Sonicatethesuspensiononiceaccordingtotheinstructionsprovidedbythe

manufacturer(e.g.,use10x15sburstswitha15scoolingperiodbetweeneach burst).

• Carefully check samples´ appearance after sonication. If the lysate is still viscousfromincompletefragmentationofDNA,add5μg/mLDNaseIandstir on ice for 15 min.

4 Clarify lysate

• Centrifuge the crude lysate at 10,000 x gfor30minat4 °Ctoremovecellulardebris.

• Carefully transfer the supernatant to a clean tube without disturbing thepellet.Ifthesupernatantisnotclear,centrifugeasecondtimeorfilterthrougha 0.45 μm membrane (e.g., cellulose acetate).

• Storesupernatantonice.

11MACHEREY-NAGEL – 05 / 2013, Rev. 04


Proceed to section4.2(BatchpurificationofGST-taggedproteins),4.3(Batch/gravity-flowpurificationofGST-taggedproteins),4.4(SpincolumnpurificationofGST-taggedproteins),or4.5 (FPLC™purificationofGST-taggedproteins).


4.2 Batch purification of GST-tagged proteins

Protino®GlutathioneAgarose4Bissuppliedas75%suspension.Forbatchpurificationof GST-tagged proteins, the original 75% suspension can be used directly afterperforming thenecessaryequilibrationsteps (refer tostep1a).Alternatively,a50%pre-equilibratedworkingsuspensioncanbeproduced(refertostep1b)thatmaybeuseddirectlyormaybestoredat4°Cforupto1monthandusedifrequired(refertostep 1c).

1a Equilibration (start with the 75 % original suspension)

• Determine the bed volume of Protino®GlutathioneAgarose4Brequiredfor your application (see section 2.2).

• Resuspend Protino® GlutathioneAgarose 4B bymixing thoroughly toachieve a homogeneous suspension.

• Immediatelytransfersufficientsuspensiontoanappropriatetube.Pipette1.333mLoftheoriginal75%suspensionpermLofbedvolumerequired.

• Sediment the gel by centrifugation at 500 x g for 2–5 min. Carefully decant the supernatant (storage solution) and discard it.

• Add10bedvolumesofPBStoequilibratethegel.Inverttomix.• Sediment the gel by centrifugation at 500 x g for 2–5 min. Carefully

decant the supernatant and discard it.• Proceed to step 2.

1b Preparation of a 50 % pre-equilibrated suspension (optional)

Protino® GlutathioneAgarose 4B is supplied as 75 % suspension. Itmay beadvantageous to prepare and store pre-equilibrated Protino® GlutathioneAgarose 4B suspension, e.g. when several preparations are performed inparallelorwhendailyexperimentsareplanned.Thefollowingstepsproducea50 %suspensionofpre-equilibratedProtino®GlutathioneAgarose4Bwhichmaybe used directly or may be stored at 4 °Cforupto1monthandusedifrequired(refer to section 1c).



• Immediatelytransfersufficientsuspensiontoanappropriatetube.Pipette1.333mLoftheoriginal75 %suspensionpermLofbedvolumerequired.

• Sediment the gel by centrifugation at 500 x g for 2–5min. Carefullydecant the supernatant (storage solution) and discard it.

• Add10bedvolumesofPBStoequilibratethegel.Inverttomix.• Sediment the gel by centrifugation at 500 x g for 2–5min. Carefully

decant the supernatant and discard it.



• Add1bedvolumeofPBS.Inverttomix.Thisresultsina50 %suspensionof Protino®GlutathioneAgarose4B,whichmaybeuseddirectlyormaybe stored at 4 °C for up to 1 month.

• If the suspension is used directly proceed to step 1c.

1c Start with 50 % pre-equilibrated suspension

• Prepare 50 %pre-equilibratedsuspensionaccordingtosection1b.• Determine the bed volume of Protino® GlutathioneAgarose4Brequired

for your application (see section 2.2).• Resuspend Protino® GlutathioneAgarose 4B bymixing thoroughly to

achieve a homogeneous suspension.• Immediately transfer sufficient suspension to an appropriate tube.

Pipette 2 mL of the 50 % pre-equilibrated suspension per mL of bedvolumerequired.

• Proceed to step 2.

2 Batch binding

• AddtheclarifiedE. colilysatetotheequilibratedgel.• Mixthesuspensiongentlyfor30minatroomtemperature.• Sediment the gel by centrifugation at 500 x g for 2–5 min. Carefully

decant the supernatant and discard it.

3 Washing

• Washthegelbyadding10bedvolumesofPBS.Inverttomix.• Sediment the gel by centrifugation at 500 x g for 2–5 min. Carefully

decant the supernatant and discard it.• Repeatthewashingsteptwice(totalwash3x10bedvolumesofPBS).

4 Elution

• Add 1 bed volume of Elution Buffer to the sedimented gel.• Mixthesuspensiongentlyfor10minatroomtemperaturetoliberatethe

GST-taggedproteinfromthegel.• Sediment the gel by centrifugation at 500 x g for 2–5 min. Carefully

decantorpipettethesupernatantinanewtubeandstoreonice.• Repeattheelutionsteptwice.Poolthecollectedeluates.

Note: The amount of elution buffer required may vary among fusion proteins. Volumes may have to be increased or additional elution steps may be necessary. Use a Bradford protein assay, SDS-PAGE or measure the absorbance at 280 nm to determine the yield of the eluted GST-tagged protein.



4.3 Batch / gravity-flow purification of GST-tagged proteins

1 Equilibration



• Immediately transfer the determined volume of suspension to an appropriate chromatography column (e.g., Protino® Columns 14 mL, 35mL;seeorderinginformation).Pipette1.333mLoftheoriginal75%suspensionpermLofbedvolumerequired.

• Allowthecolumntodrainbygravity.• Toequilibratethegel,add10bedvolumesofPBS.Allowthecolumnto

drain by gravity. Avoid disturbing the resin.

2 Batch binding

• ClosecolumnoutletwithcaporParafilm®.• AddclarifiedE. colilysatetothegelandclosecolumninletwithcapor

Parafilm®.• Mixthesuspensiongentlyfor30minatroomtemperature.• Removebottomandtopcapandallowthecolumntodrainbygravity.

3 Washing

• Towashthegel,add10bedvolumesofPBS.Allowthecolumntodrainby gravity. Avoid disturbing the resin.

• Repeatthewashingsteptwice(totalwash3x10bedvolumesofPBS).

4 Elution

• Closecolumnoutletwithcapandadd1bedvolumeofElutionBuffer.• Close column inlet and mix the suspension gently for 10 min at room

temperaturetoliberatetheGST-taggedproteinfromthegel.• Remove bottom cap and collect the eluate.• Repeattheelutionsteptwice.Poolthecollectedeluates.




4.4 Spin column purification of GST-tagged proteins

Inthisexemplaryprotocolmini-spincolumns(ReceiverColumns20μm, see ordering information,section6.3)with50μL Protino®GlutathioneAgarose4Bbedvolumeareused to purify up to 400 μgofGST-taggedprotein.

1 Equilibration


• Immediatelytransfer67μLoftheoriginal75%suspensiontoaReceiverColumnplacedinacollectingtube(67μLoftheoriginal75%suspensioncorresponds to 50 μL of bed volume).

• Centrifuge at 500 x g for 30 s.• Toequilibratethegeladd500μLofPBS.• Centrifuge at 500 x gfor30s.Discardflowthrough.

2 Batch binding

• Closecolumnoutletwithcap.• Addupto700μLofclarifiedE. coli lysate to the gel and close the lid.• Mixthesuspensiongentlyfor30minatroomtemperature,e.g.,usingan

eppendorfThermomixer.• Remove bottom cap and place Receiver Column in a collecting tube.• Centrifuge at 500 x gfor30s.Discardflowthrough.

3 Washing

• Towashthegel,add500μLofPBS.• Centrifugeat500xgfor30s.Discardflowthrough.• Repeatthewashingsteptwice(totalwash3x500μLofPBS).Discard

flowthroughbetweenwashingsteps.




4 Elution

• Closecolumnoutletwithcap.Add50μL of Elution Buffer and close the lid.

• Mixthesuspensiongentlyfor10minatroomtemperaturetoliberatetheGST-taggedproteinfromthegel.

• RemovebottomcapandplaceReceiverColumnina1.5or2mLmicro-centrifuge tube.

• Centrifuge at 500 x g for 30 s.• Repeattheelutionsteptwice.Poolthecollectedeluates.




4.5 FPLC™ purification of GST-tagged proteins

Protino®GlutathioneAgarose4B,withamaximumflowrateofapproximately250cm/h,is compatible with common low pressure chromatography columns and FPLC™applications. We recommend columns equipped with an adjustable plunger/flowadapter.BindingkineticsbetweenGSTandimmobilizedglutathioneisrelativelyslow.Therefore use low rates for the loading step to allowmaximal binding of theGST-taggedprotein.Theflowrateforequilibration,washing,andelutioncanbeincreasedtoreducethepurificationtime(seeTable2).

1 Preparing the chromatography system

• PurgethepumpwithPBS.Assurethatallairisdisplaced.• Determine the bed volume of Protino® Glutathione Agarose 4B

required for your application (see section 2.2). Choose a appropriatechromatographycolumn(e.g.,fromOmnifitorGEHealthcare).Ifmorethan75%ofthecolumnvolumeistobepacked,equipthecolumnwithan extension to hold the complete volume of the agarose suspension.

• Eliminate air from outlet tubing and end piece of the column by injecting PBSintooutlettubing.Closeoutletofcolumn.Leave~1cmofbufferabove the support net or frit.

• InjectPBSintotheinlettubingoftheupperplungertoeliminateair.PlaceplungerintoabeakercontainingPBSuntiluse.

2 Column packing

• Resuspend Protino® Glutathione Agarose 4B by mixing thoroughlyto achieve a homogeneous suspension. Immediately transfer the determined volume of suspension to an appropriate vacuum flask.Pipette1.333mLoftheoriginal75%suspensionpermLofbedvolumerequiredandde-gas.

• Pour the entire slurry into the column in one continuous motion along a glassrodheldagainsttheinnerwallofthecolumn.

• Carefully fill the remaining space with PBS. Insert the upper plungerintothecolumnwithoutintroducingairbubbles.Connecttheinletofthecolumn to a pump.

• Open the column outlet and start the pump. Pass PBS through thecolumnatapackingflowrateofapproximately250cm/h(seeTable2below) until height of gel bed becomes constant.Stop the pumpandclose the column outlet.

• Position the upper plunger on top of the column bed. Avoid to introduce airbubbles.Openthecolumnoutletandstartthepumpataflowrateofapproximately250cm/huntilthebedisstable.Re-positiontheplungeron the medium surface as necessary.



3 Column equilibration

• Equilibrate the columnwith approximately 5–10 bed volumes of PBSuntil the baseline at 280 nm is stable.

4 Binding

• Loadthecentrifugedorfilteredsampleontothecolumn.

NOTE: Binding kinetics between GST and immobilized glutathione is relatively slow. Therefore use low rates for the loading step to allow maximal binding of the GST-tagged protein.

• Collectflowthroughandanalyze,e.g.,bySDS-PAGEtoverifythattheGST-tagged protein has bound. If the fusion protein is found in earlyfractionsof theflow-through, theflowrateshouldbedecreased. If thefusion protein is absent in early fractions and does appear in late fractions oftheflow-throughthecolumncapacityhasbeenexceeded.Inthiscaseprotein load should be reduced or bed volume should be increased.

5 Washing

• Washthecolumnwith10bedvolumesofPBSoruntil thebaselineat280 nm is stable.

6 Elution

• ElutetheGST-taggedproteinwith10bedvolumesofElutionBufferandcollect fractions.

• UseaBradfordproteinassay,SDS-PAGE,ormeasuretheabsorbanceat280nmtoidentifythefraction(s)whichcontain(s)themajorityoftheelutedGST-taggedproteinandanalyzebySDS-PAGE.

Table 2: Recommended flow rates for Protino® Glutathione Agarose 4B

Column diameter

[mm]

Bed volume [mL]

Packing Equilibration Washing Elution

Binding

Linearflowrate[cm/h]1≤250 ≤180 ≤180

Volumetricflowrate[mL/min]6.6 1 1.4 1 0.3–116 10 7 5 0.5–5

1 Forconvertingfromlineartovolumetricflowrateseesection6.2.



5 Regeneration and storageReuse of Protino®GlutathioneAgarose 4B should only be performedwith identicalGST-taggedproteinstoavoidpossiblecross-contamination.Thelifetimeofthematrixdepends on the nature of the sample.

IfasingleGST-taggedproteinistobepurifiedseveraltimes,simplywashwith10bedvolumesofPBS.

Basic cleaning:Washresinwithapproximately10bedvolumesof100mMTris-HCl+0.5MNaCl,pH8.5,followedbyapproximately10bedvolumesof100mMsodiumacetate+0.5MNaCl,pH4.5.Repeattheabovewashcyclestwice.Washwith5bedvolumesofPBS.

Rigorous cleaning: Toremoveprecipitatedordenaturedproteins,washwith2bedvolumesof6Mguanidinehydrochloride,immediatelyfollowedby5–10bedvolumesofPBS.Toremovehydrophobicallyboundcontaminants,washwith4bedvolumesof70%ethanolor1%TritonX-100followedby5–10bedvolumesofPBS.

Ifyouwillnotbeusingthematriximmediately,washwithadditional5bedvolumesof20%ethanolandstoreat4°C.



6 Appendix

6.1 Troubleshooting

Problem Possible cause and suggestions

Lowproteinyield

Problems with vector construction • Ensure that protein and tag are in frame.

Low protein expression• Optimize bacterial expression conditions.

Fusion protein forms insoluble aggregates (inclusion bodies)• Lowerthegrowthtemperaturefrom37 °Cto30–15 °C.

Extraction may be insufficient• Check extraction conditions (lysozyme, sonication).

• Useupto2 %ofanon-ionicdetergenttoimprovecelldisruptionand/or solubilization of the fusion protein.

Fusion protein does not bind efficiently

Sonication may have been to severe• Choosemildersonicationconditions.Over-sonicationcanalter

the conformation of theGSTmoiety and prevents the fusionprotein from binding to Protino®GlutathioneAgarose4B.

Reducing agent missing• AddingDTTtothelysisbuffer(finalconcentration5mM)prior

to cell lysis can significantly increase binding of some fusionproteins.

Flow rate too high• Decreaseflowratefortheloadingsteptoallowmaximalbinding

oftheGST-taggedprotein.

Concentration of fusion protein is too dilute• Concentrate the sample. Yield depends on the concentration of

the fusion protein in the lysate. If the sample is too dilute, fusion proteinsmaynotbindefficiently.

21




Fusion protein does not bind efficiently (continued)

Protino® Glutathione Agarose 4B has been used several times• Clean matrix according to section 5 or use fresh matrix.

Immobilized glutathione can be degraded by γ-glutamyltranspeptidase activity in E. colicelllysates.Therefore,matriceswithimmobilizedglutathionehaveafinitelifetime.

Fusion protein elutes inefficiently

Low elution volume • Increase the volume of Elution Buffer. Depending on the nature

of the fusion protein and the amount of protein loaded, additional elutionstepsorbuffervolumeisrequired.

Flow rate too high• Decreaseflowrateduringelution.

Incorrect buffer composition• Check composition and pH of the Elution Buffer. In some cases

up to 50mM reduced glutathione may be used to improveelution.

Elution Buffer not prepared immediately before use• Prepare Elution Buffer immediately before use.

Poor protein purity

Insufficient washing• IncreasethenumberofwasheswithPBS.

Degradation of GST fusion protein• Addaproteaseinhibitortothelysissolution.Multiplebandsmay

be the result of partial degradation by host proteases during the purificationprocedure.

• Keep all samples and buffers on ice to reduce the activity of proteases.

• Useaprotease-deficienthost.Multiplebandsmaybetheresultofpartialdegradationbyhostproteasesduringcellgrowth.

Sonication may have been too severe• Choosemildersonicationconditions.Over-sonicationcanlead

to the co-purification of host proteins with the GST-taggedprotein.




Poor protein purity(continued)

Co-purification of chaperonins• Several chaperonins, that are involved in protein folding,

may co-purify with GST fusion proteins, for example, DnaK(~70kDa),DnaJ(~37kDa),GrpE(~40kDa),GroEL(~57kDa),GrpE (~40kDa),GroEL (57kDa),GroES (~10kDa).Severaladditionalpurificationstepshavebeendescribed.Forexampleco-purificationofDnaKcanbeavoidedbytreatingthecelllysatewith5mMMgCl2and5mMATPpriortopurification.DnaKcanbedissociatedfromothercomponentsinthepresenceofATPandMg2+.

6.2 Converting from linear to volumetric flow rates and vice versa

Converting from linear flow rate [cm/h] to volumetric flow rate [mL/min]

VF [mL/min]=LF[cm/h]

x A [cm2]=LF[cm/h]

xπx(d[cm])2

60 60 4

Converting from volumetric flow rate [mL/min] to linear flow rate [cm/h]

LF [cm/h]=VF[mL/min]x60

=VF[mL/min]x60x4

A [cm2] πx(d[cm])2

LF Linearflowrate[cm/h]VF Volumetricflowrate[mL/min]A Columncross-sectionalarea[cm2]d Columninnerdiameter[cm]

23



6.3 Ordering information

Product REF Pack of

Protino®GlutathioneAgarose4B 745500

745500

.10

.100

10 mL (settled agarose beads)

100 mL(settled agarose beads)

Protino®GST / 4BColumns1mL 745510.5 5 columns

Protino®GST / 4BColumns5mL 745515 745515

.1

.51 column 5 columns

Receiver Columns 20 μm 740522. 740522. 740522.

10 50 250

10 columns 50 columns

250 columns

Protino® Columns 14 mL(withoutcaps)

745250.10 10 columns

Protino® Columns 35 mL(withoutcaps)

745255.10 10 columns

Visit www.mn-net.com for more detailed product information.



6.4 Product use restriction / warranty

Protino® Glutathione Agarose 4B products are intended, developed, designed, and soldFORRESEARCHPURPOSESONLY,except,however,anyotherfunctionoftheproductbeingexpresslydescribedinoriginalMACHEREY-NAGELproductleaflets.

MACHEREY-NAGEL products are intended for GENERAL LABORATORY USEONLY!MACHEREY-NAGELproductsaresuitedforQUALIFIEDPERSONNELONLY!MACHEREY-NAGEL products shall in any event only be used wearing adequatePROTECTIVE CLOTHING. For detailed information please refer to the respectiveMaterial Safety Data Sheet of the product! MACHEREY-NAGEL products shallexclusivelybeused inanADEQUATETESTENVIRONMENT.MACHEREY-NAGELdoes not assume any responsibility for damages due to improper application of our products inother fieldsofapplication.Applicationon thehumanbody isSTRICTLYFORBIDDEN.Therespectiveuserisliableforanyandalldamagesresultingfromsuchapplication.

DNA/RNA/PROTEIN purification products of MACHEREY-NAGEL are suitable forIN VITRO-USESONLY!

ONLY MACHEREY-NAGEL products specially labeled as IVD are also suitablefor IN VITRO-diagnostic use. Please pay attention to the package of the product.IN VITRO-diagnosticproductsareexpresslymarkedasIVDonthepackaging.

IF THERE IS NO IVD SIGN, THE PRODUCT SHALL NOT BE SUITABLE FORIN VITRO-DIAGNOSTICUSE!

ALL OTHER PRODUCTS NOT LABELED AS IVD ARE NOT SUITED FOR ANYCLINICALUSE(INCLUDING,BUTNOTLIMITEDTODIAGNOSTIC,THERAPEUTICAND/ORPROGNOSTICUSE).

Noclaimor representations is intended for itsuse to identifyanyspecificorganismor for clinical use (included, but not limited to diagnostic, prognostic, therapeutic, or bloodbanking).Itisratherintheresponsibilityoftheuseror-inanycaseofresaleoftheproducts-intheresponsibilityoftheresellertoinspectandassuretheuseoftheDNA/RNA/proteinpurificationproductsofMACHEREY-NAGELforawell-definedandspecificapplication.

MACHEREY-NAGELshallonlyberesponsiblefortheproductspecificationsandtheperformancerangeofMNproductsaccordingtothespecificationsofin-housequalitycontrol, product documentation and marketing material.

ThisMACHEREY-NAGELproductisshippedwithdocumentationstatingspecificationsand other technical information. MACHEREY-NAGEL warrants to meet the statedspecifications.MACHEREY-NAGEL´ssoleobligationandthecustomer´ssoleremedyis limited to replacement of products free of charge in the event products fail to perform aswarranted.Supplementary reference ismade to thegeneralbusiness termsandconditionsofMACHEREY-NAGEL,whichareprintedonthepricelist.Pleasecontactusifyouwishtogetanextracopy.

ThereisnowarrantyforandMACHEREY-NAGELisnotliablefordamagesordefectsarising in shipping and handling (transport insurance for customers excluded), or out of accident or improper or abnormal use of this product; defects in products or

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components not manufactured byMACHEREY-NAGEL, or damages resulting fromsuchnon-MACHEREY-NAGELcomponentsorproducts.

MACHEREY-NAGEL makes no other warranty of any kind whatsoever, andSPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OFANY KINDORNATUREWHATSOEVER, DIRECTLYOR INDIRECTLY, EXPRESSOR IMPLIED, INCLUDING, WITHOUT LIMITATION, AS TO THE SUITABILITY,REPRODUCTIVITY, DURABILITY, FITNESS FORA PARTICULAR PURPOSE ORUSE,MERCHANTABILITY,CONDITION,ORANYOTHERMATTERWITHRESPECTTOMACHEREY-NAGELPRODUCTS.

In no event shall MACHEREY-NAGEL be liable for claims for any other damages,whether direct, indirect, incidental, compensatory, foreseeable, consequential, orspecial(includingbutnotlimitedtolossofuse,revenueorprofit),whetherbaseduponwarranty,contract,tort(includingnegligence)orstrictliabilityarisinginconnectionwiththesaleorthefailureofMACHEREY-NAGELproductstoperforminaccordancewiththestatedspecifications.ThiswarrantyisexclusiveandMACHEREY-NAGELmakesnootherwarrantyexpressedorimplied.

The warranty provided herein and the data, specifications and descriptions of thisMACHEREY-NAGELproductappearinginMACHEREY-NAGELpublishedcataloguesand product literature are MACHEREY-NAGEL´s sole representations concerningtheproductandwarranty.Nootherstatementsorrepresentations,writtenororal,byMACHEREY-NAGEL´semployees,agentorrepresentatives,exceptwrittenstatementssignedbyadulyauthorizedofficerofMACHEREY-NAGELareauthorized;theyshouldnot be relied upon by the customer and are not a part of the contract of sale or of this warranty.

Productclaimsaresubjecttochange.ThereforepleasecontactourTechnicalServiceTeam for the most up-to-date information on MACHEREY-NAGEL products. Youmayalsocontactyour localdistributor forgeneralscientific information.Applicationsmentioned inMACHEREY-NAGEL literatureareprovided for informationalpurposesonly.MACHEREY-NAGELdoesnotwarrantthatallapplicationshavebeentestedinMACHEREY-NAGELlaboratoriesusingMACHEREY-NAGELproducts.MACHEREY-NAGELdoesnotwarrantthecorrectnessofanyofthoseapplications.

Lastupdated:07/2010,Rev.03

Please contact: MACHEREY-NAGELGmbH&Co.KG Tel.:+492421969-270 [email protected]

Trademarks:ÄKTAdesigandFPLCaretrademarksofGEHealthcarecompaniesBioLogicandProfiniaaretrademarksofBIO-RADLaboratories,Inc.ParafilmisaregisteredtrademarkofBemisCompany,Inc.ProtinoisaregisteredtrademarkofMACHEREY-NAGELAll used names and denotations can be brands, trademarks, or registered labels of their respective owner–alsoiftheyarenotspecialdenotation.Tomentionproductsandbrandsisonlyakindofinformation (i.e., it does not offend against trademarks and brands and can not be seen as a kind ofrecommendationorassessment).Regardingtheseproductsorserviceswecannotgrantanyguaranteesregardingselection,efficiency,oroperation.

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