407PR

G-Biosciences 1-800-628-7730 1-314-991-6034 technical@GBiosciences.com

AGenoTechnology,Inc.(USA)brandname

Protein Purification
STRATEGY
For Optimizing Protein Purification

think proteins! think G-Biosciences

www.GBiosciences.com

INTRODUCTION.................................................................................................................4
ITEM(S)SUPPLIED(CAT.#786184)..................................................................................4
IMPORTANTINFORMATION..............................................................................................5
PROTEINACTIVITYASSAY..............................................................................................5
PROTEASEACTIVITY.......................................................................................................5
PROTEINESTIMATIONASSAY........................................................................................5
ELECTROPHORESIS.........................................................................................................5
PREPARATIONOFPROTEINEXTRACT............................................................................5
ANALYSISOFPROTEINPURIFICATION...........................................................................6
PROTOCOL:PROTEINEXTRACTION...................................................................................6
PROTOCOL:PURIFICATION................................................................................................7
I.FRACTIONATIONOFPROTEINBYLOWERINGTHEPH................................................7
PRINCIPLE......................................................................................................................7
ITEM(S)REQUIRED.........................................................................................................7
ADDITIONALITEMSREQUIRED......................................................................................7
PROTOCOL.....................................................................................................................7
BUFFEREXCHANGE&EQUILIBRATIONOFTHEFRACTIONS..........................................8
RESULTS&CONCLUSIONS.............................................................................................8
II.AMMONIUMSULFATEFRACTIONATION..................................................................9
PRINCIPLE......................................................................................................................9
ITEM(S)REQUIRED.........................................................................................................9
ADDITIONALITEMSREQUIRED......................................................................................9
PROTOCOL.....................................................................................................................9
BUFFEREXCHANGE&EQUILIBRATIONOFTHEFRACTIONS........................................10
RESULTS&CONCLUSIONS...........................................................................................10
III.HYDROPHOBICCHROMATOGRAPHY.....................................................................12
PRINCIPLE....................................................................................................................12
ITEM(S)REQUIRED.......................................................................................................12
ADDITIONALITEMSREQUIRED....................................................................................12
PROTOCOL...................................................................................................................12

Page2of20

TROUBLESHOOTING....................................................................................................14
RESULTS&CONCLUSIONS...........................................................................................14
IV.IONEXCHANGECHROMATOGRAPHY(IEC).............................................................15
PRINCIPLE....................................................................................................................15
ITEM(S)REQUIRED.......................................................................................................15
PROTOCOL...................................................................................................................15
TROUBLESHOOTING....................................................................................................17
RESULTS&CONCLUSIONS...........................................................................................17
FINALNOTES....................................................................................................................18
ADDITIONALSUPPLIES.....................................................................................................18
RELATEDPRODUCTS........................................................................................................19

Page3of20

INTRODUCTION
Proteinpurificationofanovelorunknownproteinisoftenadifficultprocessasthe
physicalpropertiesoftheproteinareunknown.Mostproteinpurificationprotocols
involvemorethanonepurificationmethodandthemethodsroutinelyusethephysical
propertiesoftheprotein.

TheProteinPurificationSTRATEGY kitisdesignedtoallowresearchertheopportunity
toquicklyestablishasuitableproteinpurificationprotocol.TheSTRATEGYkituses
differentextractionandpurificationtechniques,allowingtheresearchertodetermine
theirbestprotocol,withouthavingtowastetimeormoneyonarangeofpurification
kits.Whencarefullyfollowed,proteinpurificationstrategycanbeestablishedwithina
fewdays.
ITEM(S) SUPPLIED (CAT. # 786184)
Description

Size

AcidPrecipitationBufferI

1ml

AcidPrecipitationBufferII

1ml

AmmoniumSulfateSolution

12ml

AnionicResinColumn

3columns

CationicResinColumn

3columns

HPElutionBuffer

50ml

HPLoadingBuffer

50ml

LoadingBufferI

50ml

LoadingBufferII

50ml

LoadingBufferIII

50ml

PhenylHPColumn

2columns

SodiumChloride(4M)

SpinOUT GT600,1ml

50ml
10/bag

Page4of20

IMPORTANT INFORMATION
ProteinActivityAssay
Beforestartingthepurificationofaprotein,theproceduresforidentifyingorassaying
theproteinsfortheirbiologicalactivitymustbeestablished.
ProteaseActivity
Ithasbeenwidelyrecognizedthatendogenousproteaseactivitymaydamagethe
proteinsduringpurificationprocedures.Inhibitionofproteaseactivitymayimprovethe
recoveryofproteintobepurified.Awidespectrumproteaseinhibitorcocktailis
generallypreferred.Itishighlyrecommendedthataproteaseinhibitorcocktailbe
addedtotheproteinextractionbuffer.Wesupplyacocktailofproteaseinhibitors

(ProteaseArrest ,Cat.#786108)forinhibitingproteaseactivityduringpurification.

ProteaseArrest isanexcellentinhibitorofserine,cysteineandmetalloproteasesand
thereforesuitablefortheprotectionofproteinduringpurification.
ProteinEstimationAssay
Areliableandreproduciblemethodofproteinestimationmustbeused.Several
methodsareavailable;however,amethodthatisfreefrominterferencefrom
detergents,dye,reducingagents,andvariousothercommonlyusedagentsmustbe

used.WerecommendGBiosciencesNonInterferingProteinAssay (NI ProteinAssay,

Cat.#786005)forinterferencefreeproteinassay.
Electrophoresis
Electrophoresisisthemostwidelyusedtechniqueforproteinanalysis.Themost
commontypesofelectrophoresisare(1)denaturingelectrophoresispreformedafter
denaturingtheproteinsamplewithSDSandareducingagentand(2)nondenaturing
electrophoresis.Thenondenaturingelectrophoresisallowstheproteintomaintainits
biologicalfunction,structureandaswellasinteractionswithotherprotein(subunit
species)andnucleicacidmolecules.Youmustfamiliarizeyourselfwithelectrophoresis
techniques.
PreparationofProteinExtract
Thefirstimportantconsiderationisthepreparationofproteinextractfromwhichthe
proteinofinterestistobepurified.Theextractionbuffermustbeabletomaintainthe
biologicalactivityandintegrityoftheprotein.TheExtractionBuffermustalsobe
compatiblewithdownstreampurificationprocedures.
GBiosciencesofferawideselectionofextractionbuffers.ProteinExtractionandLysis

Buffers(PELB )areaseriesofbuffersdesignedfortheextractionofawidevarietyof
proteinfrombacterial,yeast,animalcellsandtissues.ThepHofthebuffersisnear

physiological.ThePELB buffersprovideamildenvironmentforawidevarietyof
proteinsfrombacteria,yeast,andanimalcellsandtissues.Anyadditionalagentneeded

canbeeasilyaddedintothebuffer.TheuseofPELB bufferwillsimplifythetaskof
Page5of20

optimizingpurificationprocedureand,therefore,ishighlyrecommended.Thefollowing

PELB buffersareoffered.
BacterialPELB(Cat.#786177),YeastPELB(Cat.#786179),MammalianCellPELB
(Cat.#786180),andTissuePELB(Cat.#786181).ForinformationonthePELB
seriesofextractionbuffers,pleasevisitourwebsiteatwww.GBiosciences.com.
AnalysisofProteinPurification
Ateachstepofthepurificationprocedure,theproteinmustbecarefullyanalyzedto
assessthedegreeofenrichmentandtherecoveryoftheproteinofinterest.The
followingroutineanalysisisrecommended:
1.
2.
3.

Proteinconcentrationtomonitorproteinrecovery
Gelelectrophoresisfordistributioncharacterizationaswellasidentification
oftheproteininafraction,
Proteinactivityandspecificactivity.

PROTOCOL: PROTEIN EXTRACTION

1. Selectanduseanappropriateextractionbuffertoextractproteinfromthetest
samples(cellsandtissues).
2. Prepare510mlofproteinextractstoperformtheentireprocedure.Alwaysuse
freshlypreparedproteinextracts.Theextractshouldpreferablycontain550mg
protein/ml.
3. Afterextraction,lysis,orhomogenization,centrifugeat30,000xgat4Cfor3040
minutes.
4. Collecttheclearsupernatanttoaclean,labeledtube.Thisisthecrudeprotein
extractcontainingsolubleprotein.
5. Thesedimentisinsolubledebrisandmaycontainunbrokencells,cellorganelles,
andmembraneboundproteins.Sedimentormembraneboundproteinmaybe
furthersolubilizedandextractedusingavarietyofdetergents.
6. PerformProteinPurificationAnalysisontheCrudeExtract.
a. Determinetheproteinconcentration(mgprotein/ml)
b. Analyzebyproteinelectrophoresis

Page6of20

PROTOCOL: PURIFICATION
Purificationisastepbystepprocedureforremovingimpuritiesandenrichingthe
samplewithrespecttotheproteinofinterest.Thefollowingproceduresmustbecarried
outintheorderrecommended.Afterafirstrun,theorderofpurificationmethodsmay
berearrangedtosuityourparticularneeds.
I. FRACTIONATION OF PROTEIN BY LOWERING THE PH
Principle
ProteinshavethelowestsolubilityattheirisoelectricpH(pI).WhenloweringthepHof
theextractyouwouldexpectsomeproteinintheextracttoreachtheirpIandthus
precipitateoutofthesolutiongivinganeffectivefractionationstep.Theprecipitated
proteinmightincludetheproteinofyourinterest.Themethodisalsoreferredtoasacid
precipitationandfractionation.
Item(s)Required
Description

Size

AcidPrecipitationBufferI,pH5.0

1ml

AcidPrecipitationBufferII,pH6.0

1ml

SpinOUTGT600Columns(Micro)

Additionalitemsrequired

Centrifuge

Tubes

Extractionbuffers
Protocol
1. Add100200lAcidPrecipitationBufferIand100200lAcidPrecipitationBufferII
totwoseparate1.52mlcentrifugetubes.
2. AddanequalvolumeofCrudeExtracttoeachoftheabovetubes.
3. Invertthetubeafewtimesandincubatefor5minutesonice.
4. Centrifugeat15,000xgfor5minutesat4C.
5. Collectclearlysateincleantubes,andmarkthetubesasfollows:
a. LabelthetubeAPFraction1,pH5.0
b. LabelthetubeAPFraction2,pH6.0
NOTE:Itispossiblethattheproteinofyourinterestmightprecipitateduringthe
treatmentwithAcidPrecipitationBuffers.Insuchcases,youmayrecoveryour
proteinfromthepellets.
6. WashthepelletonetimewithExtractionBuffer.Add0.5mlExtractionBuffertothe
pelletandthenremovetheextractionbufferafter510seconds.
7. Suspendthepelletin100200lofExtractionBuffer.Useapipettetiptoresuspend
thepelletandallow510minutesforthepellettodissolve.
Page7of20

8.

Centrifugethesuspensionat15,000xgfor15minutesat4Candcollecttheclear
solution.
9. LabelthetubeAPFraction3,pH5.0
10. LabelthetubeAPFraction4,pH6.0
Bufferexchange&equilibrationofthefractions

1. PrepareaSpinOUT columnforeachfractionbyremovingthetopandthen
bottomcaps.Placeintoanappropriatecollectiontube.
2.

Markonesideofthecolumnandensureinallcentrifugationsthemarkisfacing
outwardsduringcentrifugation.

3.

Centrifugethecolumnat1,000gfor2minutestoremovethestoragebuffer.This
compactstheresinandremovesthestoragebuffer.

4.

Placethecolumninanewcollectiontubeandremovethecap.

5.

Add0.5mlExtractionBuffertobeexchangedintotothecolumn

6.

Centrifugethecolumnat1,000gfor2minutestoremovethebuffer.

7.

Repeatsteps2and3threemoretimes,ensuringthebufferisdiscardedaftereach
centrifugation.

8.

Placethecolumninanewcollectiontubeandremovethecap.

9.

Slowly,apply75lofeachfractiontothecenteroftheSpinOUTresin.

10. Centrifugethecolumnat1,000gfor2minutestocollectthedesaltedprotein
solution.Discardthecolumn.
11. PerformProteinPurificationAnalysisontheFractions.
a. Determinetheproteinconcentration(mgprotein/ml)
b. Analyzebyproteinelectrophoresis
Results&Conclusions
Bycomparingtheresultsofacidprecipitationfractionations,itwouldbepossibleto
establishhowyourproteinbehavedataparticularpHandhowmuchimpurity,ifany,
wasremovedatanygivenpH.Youmayfindthattheproteinofinterestdidnot
precipitateandonlyimpurities(protein)wereprecipitatedandremovedfromthe
sample,allowingadegreeofenrichment.Anoppositescenarioisalsopossiblee.g.,the
proteinofinterestprecipitatesleavingbehindimpurity.Theprecipitatedproteinmaybe
latersolubilizedallowingadegreeofenrichment.Itisalsopossiblethatacid
precipitationirretrievablydamagestheproteinofyourinterestandinafinalanalysis
acidprecipitationmaynotproveasuitableprocedure.

Page8of20

II. AMMONIUM SULFATE FRACTIONATION

Principle
Inthepresenceofahighconcentrationofsalts,proteinprecipitatesoutofsolution.
Ammoniumsulfateisthemostwidelyusedsaltforsaltingoutproteinorprotein
fractionation.Differentproteinsprecipitateoutofsolutionatdifferentconcentrations
ofsalt.Thus,ammoniumsulfateprecipitationoffersasimpleandrapidmethodof
proteinfractionation.
IMPORTANT:Forammoniumsulfatefractionation,thecrudeextractortheprotein

solutionshouldpreferablycontain<1%nonionicdetergents.UseofthePELB seriesof
extractionbuffersissuitableforammoniumsulfatefractionation.
Item(s)Required
Description

Size

AmmoniumSulfateSolution(9095%) 12ml
SpinOUTGT600Columns(Micro)

AdditionalItemsRequired

Centrifuge

Tubes

Extractionbuffers
Protocol
1. Add0.5mlcrudeextracttoa2mlmicrofugetubelabeledTube1.
NOTE:Forbestresults,thecrudeextractshouldnotcontainanydetergent.
2. Slowly,add0.25mlAmmoniumsulfateinadropwisemanner.
3. Invertthetubeafewtimesandincubateonicefor5minutes.Centrifugeat
15,000xgat4Cfor5minutes.
4. TransferthesupernatanttoacleantubelabeledTube2.
5. CentrifugeTube1for510secondsandremoveanyresidualsupernatant.
6. Add0.1mlExtractionBuffertotheprecipitatedpelletinTube1.Vortextosuspend
thepellet.MarkthetubeAS(~30%)Fraction1
7. Add0.25mlAmmoniumsulfatetoTube2.Invertthetubeafewtimesandincubate
onicefor5minutes.Centrifugeat15,000xgat4Cfor5minutes.
8. TransferthesupernatanttoacleantubelabeledTube3.
9. CentrifugeTube2for510secondsandremoveanyresidualsupernatant.
10. Add0.1mlExtractionBuffertotheprecipitatedpelletinTube2.Vortextosuspend
thepellet.MarkthetubeAS(~45%)Fraction2
11. Add0.5mlAmmoniumsulfatetoTube3.Invertthetubeafewtimesandincubate
onicefor5minutes.Centrifugeat15,000xgat4Cfor5minutes.
12. TransferthesupernatanttoacleantubelabeledTube4.
13. CentrifugeTube3for510secondsandremoveanyresidualsupernatant.
Page9of20

14. Add0.1mlExtractionBuffertotheprecipitatedpelletinTube3.Vortextosuspend
thepellet.MarkthetubeAS(~60%)Fraction3
15. Add0.5mlAmmoniumsulfatetoTube4.Invertthetubeafewtimesandincubate
onicefor5minutes.Centrifugeat15,000xgat4Cfor5minutes.
16. TransferthesupernatanttoacleantubelabeledTube5.MarkthetubeAS(~68%)
Supernatant.
17. CentrifugeTube4for510secondsandremoveanyresidualsupernatant.
18. Add0.1mlExtractionBuffertotheprecipitatedpelletinTube4.Vortextosuspend
thepellet.MarkthetubeAS(~68%)Fraction4.
19. Vortexallthefractionsfor30secondsandthencentrifugeat15,000xgat4Cfor5
minutesandcollectclearsolutionsfromeachfraction.
Bufferexchange&equilibrationofthefractions

1. PrepareaSpinOUT columnforeachfractionbyremovingthetopandthen
bottomcaps.Placeintoanappropriatecollectiontube.
2.

Markonesideofthecolumnandensureinallcentrifugationsthemarkisfacing
outwardsduringcentrifugation.

3.

Centrifugethecolumnat1,000gfor2minutestoremovethestoragebuffer.This
compactstheresinandremovesthestoragebuffer.

4.

Placethecolumninanewcollectiontubeandremovethecap.

5.

Add0.5mlExtractionBuffertobeexchangedintotothecolumn

6.

Centrifugethecolumnat1,000gfor2minutestoremovethebuffer.

7.

Repeatsteps2and3threemoretimes,ensuringthebufferisdiscardedaftereach
centrifugation.

8.

Placethecolumninanewcollectiontubeandremovethecap.

9.

Slowly,apply75lofeachfractiontothecenteroftheSpinOUTresin.

10. Centrifugethecolumnat1,000gfor2minutestocollectthedesaltedprotein
solution.Discardthecolumn.
11. PerformProteinPurificationAnalysisontheFractions.
a. Determinetheproteinconcentration(mgprotein/ml)
b. Analyzebyproteinelectrophoresis
c. Analyzebiologicalactivity(Optional)
Results&Conclusions
Bycomparingtheresultsofammoniumsulfatefractionation,itispossibletodetermine
howyourproteinbehavedduringammoniumsulfatefractionation.Youmayfindthatat
acertainammoniumsulfateconcentration,theproteinofyourinterestispreferentially
precipitated,allowingeffectiveenrichmentofyourprotein.Theammoniumsulfate
fractionationmayberepeatedbymodifyingthevolumeofammoniumsulfateadded
intotheproteinsolution.Selecttherangeofammoniumsulfateconcentrationatwhich
mostofyourproteinprecipitates.
Page10of20

Theacidandammoniumsulfatefractionationtechniqueswillprovidevaluable
information.Theuseofthesemethodsmayenableseveralfoldenrichmentor
purificationoftheproteinincrudeextract.Dependingonresultseitheroneorboth
methodsmaybeused.Itispreferabletofirstuseacidprecipitation,whichmaybe
immediatelyfollowedwithammoniumsulfatefractionation.

Page11of20

III. HYDROPHOBIC CHROMATOGRAPHY

Principle
HydrophobicChromatographyisbasedonthefactthatproteinmolecules,inadditionto
theexpectedhydrophilicgroups(chargedgroups),canhaveextensivehydrophobic
regions.Thesehydrophobicregions,inmediafavoringhydrophobicinteraction(e.g.an
aqueoussolutionwithhighsaltconcentration)canbindtothehydrophobicligands
providedintheunchargedcolumnmatrix.Elutionisbroughtaboutbydecreasingthe
saltconcentrationoftheeluent.Insomecases,adecreaseofthesolventpolarityisalso
needed(e.g.,PEG,nonionicdetergents,denaturants,urea,chaotropicions).
Item(s)Required
Description
PhenylHPColumn

Size
2Columns

HPLoadingBuffer[2X]

50ml

HPElutionBuffer

50ml

AdditionalItemsRequired

Centrifuge

Tubes

Extractionbuffers
Protocol
1. Thefollowingprotocolshouldbecarriedoutatroomtemperature,ashydrophobic
interactionsareweakeratlowertemperatures.
2. Transfer0.25mlcrudeextractorpreviousfractioncontainingtheproteinofinterest
toa1.5mlcentrifugetube.Thetotalproteinconcentrationshouldbe0.40.6mg
protein.
3. Add0.25mlof[2X]HPLoadingBuffer.
NOTE:Whenworkingwithproteinswhichhaveatendencytoprecipitatein[2X]
HPLoadingBuffer,dilutetheLoadingBuffertoavoidprecipitationofprotein.Use
thesamedilutedLoadingBufferforpreparingtheElutionBuffer,asdescribed
below.
4. RemovethebottomclosureandpositionaPhenylHPColumninacollectiontube.
5. Dilute5ml2XHPLoadingBufferwith5mlExtractionBuffer
6. Equilibratethecolumnwith10mldilutedHPLoadingBuffer.Apply[1X]HPLoading
Bufferinsmallaliquots(23ml)andallowthebuffertodripandcollectinthe
collectiontube.AfterHPLoadingBufferiscompletelydrainedoutofthecolumn,
replacethebottomclosureonthecolumn.
7. Fortheelutionstep,theprotocolrequiresbriefcentrifugationofthecolumn.
Centrifugationshouldnotbetoosevereastodrythecolumn.Centrifugation
shouldbeatsuchamoderatespeed(~200300xgfor3040seconds)sothatit
Page12of20

removesonly6070%ofthebufferfromthecolumn,leavingbehindinthecolumn
3040%buffer.Ifnecessary,makeatrialrun(beforeloadingtheproteinsample)to
determineanappropriatecentrifugationcondition.Makeanoteofthe
centrifugationcondition(centrifugationspeedandduration)andusetheexact
sameconditionateachstep,unlessspecifiedotherwise.
8. TakefivemicrofugetubesandmarkthemasElutionBuffer1,2,3,4,&5.Mixas
follows:
Tube DilutedHPElution
HPLoadingBuffer
HPElution
Water
#
Buffer(ml)
[2X](ml)
Buffer(ml)
(ml)
1

ElutionBuffer1

0.4ml

0.1ml

0.5ml

ElutionBuffer2

0.3ml

0.2ml

0.5ml

ElutionBuffer3

0.2ml

0.3ml

0.5ml

ElutionBuffer4

0.1ml

0.4ml

0.5ml

ElutionBuffer5

1ml

9.

Apply(0.10.5ml)sample(containing0.20.3mgtotalprotein)onthecolumn.
Removethebottomclosureandallowthecolumntodrain.Allowthecolumnto
dripuntilthereisnobufferdrippingfromthecolumn.Incubatefor5minutesat
roomtemperature.CollecttheeluentinacollectiontubeandmarkthetubeasHP
EluentIA.
10. Positionthecolumnonacleancollectiontubeandcentrifugethecolumnforabrief
3040seconds.MarkthetubeandtheeluentasHPEluentIB.
11. Elutetheproteinwithstepgradient.StartingfromElutionBuffer1,elutethe
proteinasfollows.
a. Apply0.25mlofeachelutionbuffer(intheorderlistedbelow,oneafter
another),incubatefor5minutes.Spinthecolumnasdescribedabove(atthe
samespeedandduration),collectandmarktheeluentsasfollows:
i. Apply0.25mlofElutionBuffer1andcollectthefractionandlabelit
HPFraction1
ii. Apply0.25mlofElutionBuffer2andcollectthefractionandlabelit
HPFraction2
iii. Apply0.25mlofElutionBuffer3andcollectthefractionandlabelit
HPFraction3
iv. Apply0.25mlofElutionBuffer4andcollectthefractionandlabelit
HPFraction4
v. Apply0.25mlofElutionBuffer5andcollectthefractionandlabelit
HPFraction5
12. PerformProteinPurificationAnalysisontheFractions.
a. Determinetheproteinconcentration(mgprotein/ml)
b. Analyzebyproteinelectrophoresis
c. Analyzebiologicalactivity(Optional)
Page13of20

Troubleshooting
Iftheproteinofinterestisdetectedinthefractionselutingfromthecolumn
immediatelyafterloading,inadditiontothefractionselutedoffthecolumnwiththe
elutionbufferthentoomuchproteinisloadedonthecolumn.Cutdownthetotal
proteinorsamplevolumeloadedontothecolumn.
Results&Conclusions
BycomparingtheresultsofHPFractions,itwouldbepossibletodeterminehowyour
proteinbehavedduringHPChromatography.Whethertheproteinofinterestboundto
thecolumnandatwhatsaltconcentrationtheproteinwaselutedfromthecolumn,you
wouldbeabletofindthechromatographicandelutionconditionsforeffective
enrichmentofyourprotein.
Ifthecolumndidnotimmobilizetheproteinofyourinterest,youwouldstillbeableto
establishhowmuchimpurity(protein)wasretainedbytheHPColumn.

Page14of20

IV. ION EXCHANGE CHROMATOGRAPHY (IEC)

Principle
IonicinteractionisthebasisofproteinpurificationbytheIEC.Proteincontainsregions
ofchargedgroupsonthesurfacethatinteractwiththeionexchangegroupimmobilized
onthestationaryphase(column).Immobilizedproteinsareelutedwithasaltgradient.
Item(s)Required
Description

Size

AnionicResinColumn,1.5mlresin

3Columns

CationicResinColumn,1.5mlresin

3Columns

LoadingBufferI,0.5MTris,pH6.5,20mMNaCl

50ml

LoadingBufferII,0.5MTris,pH7.5,20mMNaCl

50ml

LoadingBufferIII,0.5MTris,pH8.5,20mMNaCl

50ml

NaCl[4M]

50ml

Protocol
NOTE:Thekitissuppliedwiththreeanionandthreecationchromatographycolumns
andthreesampleloadingbuffers.Useanyonecolumnatatimeandperform
chromatographywithanyonesampleloadingbufferatatime.Youwouldbeabletorun
threechromatographyrunswithanioncolumns,usingthreeseparateLoadingBuffers(I,
II,&III)andthreechromatographyrunswithcationcolumns,usingthreeseparate
LoadingBuffers(I,II,&III).Intotal,youwouldbeabletorunsixseparate
chromatographyruns.
1.
2.

3.

4.

Selectoneanioniccolumn(oracationiccolumn)andlabelColumn1andposition
thecolumninacollectiontube.
PrepareColumnEquilibrationBufferbycombining5mlLoadingBufferIwith5ml
ExtractionBuffer.
NOTE:TheExtractionBuffershouldcontain>10mMsalt
EquilibratetheColumn1with10mlColumnEquilibrationBuffer.Apply34ml
bufferatatimeandallowthebuffertodripuntilthecolumnisemptyofbuffer.
AfterEquilibrationBufferiscompletelydrainedoutofthecolumn,replacethe
bottomclosureonthecolumn.
Forelutionsteptheprotocolrequiresabriefcentrifugationofthecolumn.
Centrifugationshouldnotbetooseveretodrythecolumn.Centrifugationshould
beatsuchamoderatespeed(~200300xgfor3040seconds)thatitremovesonly
6070%ofthebufferfromthecolumn,leavingbehindinthecolumn3040%buffer.
Ifnecessary,makeatrialrun(beforeloadingtheproteinsample)todeterminean
appropriatecentrifugationcondition.Makeanoteofthecentrifugationconditions,

Page15of20

5.

centrifugationspeedandduration,andusethesameconditionateachstep,unless
specifiedotherwise.
MixLoadingBuffer1withincreasingamountsof4MNaCl,asfollows:
Tube# ElutionBuffer# LoadingBufferI(l) NaCl[4M](l)

FinalNaCl
(mM)

ElutionBuffer1

897

13

50

ElutionBuffer2

975

25

100

ElutionBuffer3

950

50

200

ElutionBuffer4

925

75

300

ElutionBuffer5

900

100

400

ElutionBuffer6

850

150

600

ElutionBuffer7

750

250

1000

6.

Forthebestresult,usethecrudeextractthathasbeensubjectedtoammonium
sulfatefractionation,asdescribedabove.Thesamplesmustbefirstdialyzed34
hoursinextractionbuffer(containing>20mMNaCl)beforerunningIEC
chromatography.MixtheappropriatesamplewiththeLoadingBuffer1asfollows.
7. Mix0.25mlsamplewith0.25mlLoadingbufferI[0.5MTris,pH6.5,20mMNaCl].
8. Applythesample(0.10.5ml)(containing0.4.6mgtotalprotein)tothecolumn.
Incubatefor5minutes.
9. Removethebottomclosureandallowthecolumntodrain.Allowthecolumnto
dripuntilthereisnobufferdrippingfromthecolumn.Collecttheeluentina
collectiontubeandmarkthetubeasIEEluentIA.
10. Positionthecolumnonacleancollectiontubeandcentrifugethecolumnforabrief
3040seconds.MarkthetubeandtheeluentasIEEluentIB.
11. Elutetheproteinfromthecolumnbyapplying0.25mlofeachofthefollowing
elutionbuffers,oneafteranotherinthefollowingorderandcollecteluent,as
follows:
a. Apply0.25mlofElutionBuffer1andcollectthefractionandlabelit
IECFraction1
b. Apply0.25mlofElutionBuffer2andcollectthefractionandlabelit
IECFraction2
c. Apply0.25mlofElutionBuffer3andcollectthefractionandlabelit
IECFraction3
d. Apply0.25mlofElutionBuffer4andcollectthefractionandlabelit
IECFraction4
e. Apply0.25mlofElutionBuffer5andcollectthefractionandlabelit
IECFraction5
Page16of20

f.

Apply0.25mlofElutionBuffer6andcollectthefractionandlabelit
IECFraction6
g. Apply0.25mlofElutionBuffer7andcollectthefractionandlabelit
IECFraction7
12. PerformProteinPurificationAnalysisontheFractions.
a. Determinetheproteinconcentration(mgprotein/ml)
b. Analyzebyproteinelectrophoresis
c. Analyzebiologicalactivity(Optional)
13. Repeattheproceduredescribedabovewiththeremainingcolumns(anionicand
cationic)usingtheLoadingBufferIIandIII,respectively.Usethesameschemefor
preparingtheEquilibrationBufferandElutionBuffer.
Troubleshooting
Iftheproteinofinterestisdetectedinthefractionselutingfromthecolumn
immediatelyafterloading,inadditiontothefractionselutedoffthecolumnwiththe
elutionbufferthentoomuchproteinisloadedonthecolumn.Cutdownthetotal
proteinorsamplevolumeloadedontothecolumn.
Results&Conclusions
BycomparingtheresultsofIECFractions,with(anionicorcationic)columnsandunder
differentloadingandelutionbufferconditionsitwouldbepossibletodeterminehow
yourproteinbehavedduringIECchromatography.Youwouldbeabletofind
chromatographicloadingandelutionconditionsforobtainingeffectiveenrichmentof
yourprotein.

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FINAL NOTES
Thefractionationtechniquesoutlinedabovewillprovideresearchersinformation
necessaryfordevelopingastrategyforproteinpurification.
1.

Acidfractionationand/orammoniumsulfatefractionationshouldbeusedfirstto
fractionatethecrudeproteinextract.IfAcidPrecipitationFractionationcanbe
usedforfractionationoftheproteinofyourinterest,itshouldbeusedfirst
followedbyammoniumsulfatefractionation.

NOTE:Highspeedultracentrifugationmayalsobeusedforinitialfractionationof
proteinincrudeextract,e.g.,whenproteinsolutionorproteinextractiscentrifugedat
ultrahighgforce,thecentrifugalforceallowssedimentationofhighmol.wt.protein
molecules,cellularfractions,andcellorganelles.Itnormallyrequiresprolonged
centrifugationinrefrigeratedultracentrifuges.Ultracentrifugationmay,therefore,be
usedfortheseparationofhighmol.wt.proteinfromthelowmol.wt.proteinmolecules.
Ultracentrifugationisgenerallysuitableforseparationof>1000kdmoleculesfromlow
molecularmolecules.
2.

Afteracidand/orammoniumsulfatefractionation,followhydrophobicand/orion
exchangechromatography.Whenbothchromatographyareused,oneafter
another,itwillprovideafairlyhighdegreeofproteinpurification.

NOTE:Gelfiltrationorpermeationchromatographymayalsobeusedfortheseparation
ofhighmol.wt.proteinfromthelowmol.wt.proteinmolecules.Followingacidand/or
ammoniumsulfatefractionationandpriortorunningthehydrophobicand/orion
exchangechromatography,gelfiltrationchromatographymaybeintroducedto
separatethehighmolwt.proteinfromthelowmol.wt.proteinmolecules.Collectthe
activefractionsandthenperformHPandIECchromatography.
3.

Forevenhigherpurification,additionalaffinitybasedchromatography,gradient
ultracentrifugation,isoelectric,chromofocusing,orelectroelutiontechniques
maybeemployed.

ADDITIONAL SUPPLIES
Afteroptimizingtheproteinpurificationstrategy,thereagents,columns,andsupplies
providedinthekitmaybeorderedseparately.Inaddition,custommadecolumnsand
bufferscanalsobeobtainedforfurtherpurificationworks.

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RELATED PRODUCTS
DownloadourProteinPurificationHandbook.

http://info.gbiosciences.com/completeproteinpurificationhandbook/
Forotherrelatedproducts,visitourwebsiteatwww.GBiosciences.comorcontactus.
Lastsaved:7/24/2012CMH

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www.GBiosciences.com

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