Affinitychromatography(withHIS-taggedproteins)

ThisprotocolisdescribedinlessdetailinCopelandetal,Nature,379,162-165,1996.

Affinitychromatographycanbeperformedusinganumberofdifferentproteintags.Inourlabwehavehadsuccessusingpoly-hisitidinetaggedproteinsforcreationofourproteinaffinitycolumns.Thehistidinetagisveryshort(10hisresidues)andshouldnotaltertheconformationofthetaggedprotein,norshoulditbeinvolvedinartifactualinteractions.Thepoly-histagbindstoanickelchelateresinforcreationofthecolumn.Weusetheresinaloneforthecontrolcolumns.Mostoften,wechallengeourcolumnswith35S-labeled,invitrotranslatedproteins,however,proteinsfromavarietyofsourcescanbeusedanddetectedappropriately.

Theprotocolbelowdescribesourmethod.Theorderofthestepshasbeenoptimisedsothatboththeaffinitycolumnsandinvitrotranslatedproteinarereadyatthesametime.

1)BeginbygettingouttheDNAand35S-methioninefortheinvitrotranslationreactions.

2)Whilethesethaw,preparesolutionsforaffinitycolumns:ACBuffer:(10%glycerol,100mMNaCl,20mMTrispH7.6,0.5mMEDTA,0.1%Tween-20,ddH2O).

NOTE:Youmaywishtoaddproteaseinhibitors(eg.TPCK),butusuallywedon"t;youmayalsochoosetofiltersterilizethebuffer,butagain,usuallywedon"t.

5%milkpowdersolution:10mLACBuffer+0.5gmilkpowder.Mixonashakerplatformuntildissolved.

Storebothsolutionsonice.

4)Preparethenickelresin.50uL(settledvolume)ofresinisrequiredforeachexperimentalandcontrolcolumn.TransfertheappropriatevolumeofslurryintotwosiliconizedEppendorftubes,oneforthecontrolresin,onefortheproteincoupling.TheresinshouldbeallowedtosettleandthenwashedonceinwaterandtwiceinACbuffer.Ineachwashsteptheresincanbeallowedtosettlebygravityorbycentrifugingat1.5Kfor3minutesinabenchtopcentrifuge.

5)Betweenwashesoftheresinbegintosetuptheinvitrotranslationreactionsfortheproteinsthataretobetestedforbinding.Wefollowthestandardprotocolfora25uLreactionusingthePromegaTNTcoupledtranscription/translationkit.

6)Whilethereactionsareincubatingcoupleyourproteintotheresin.Weuse1mgofpureHis-taggedproteinpermLofresin.However,theoptimalratiomayvarydependingontheinteractionbeingpursued.Wetreatthecontrolresinwiththesamebufferthattheproteinisstoredin.Allowthecouplingtoproceedfor30minutesonice.

7)Preparespincolumnsforremovalofunicorporated35S-methioninefromthetranslationreaction.Thecolumnsaremadein1mLsyringesblockedwithglasswool.TheresinisG-25(BioRad)equilibratedinACbuffer.FollowtheManiatisprotocolforspincolumns.

NOTE:inthefollowingsteps,youwanttoavoidfoamingandbubblesasmuchaspossIBLe,asthebubblescancontributetoproteindenaturation.So,alwaysmixgently.

8)Whiletheproteinisbeingcoupled,andbetweenspinsinthepreparationofspincolumns,begintosetupthehousingsfortheaffinitycolumns.Weuse200uLtipswithbevelledends(Diamed#230-1673-03K).Thetipshavetobeblockedwithglassbeadstopreventtheresinfromleakingout.UseSigmaAcidwashed212-300micronglassbeadsthathavebeenwashedinwater.Beadsshouldbeaddedtoalevelof7mmattheendofthetip.Afterthebeadshavebeentransferred,removeexcesswaterandthenadd100uLofACbuffer.Thisistopreventthecontactofproteinresindirectlywithwater.

Atthistimethefractioncollectiontubescanalsobelabeled.Thecolumnsarerunstandingineppendorftubesonice,thecolumnsaremovedtonewtubesforeachfraction.7collectiontubeswillbeneededforeachcolumn:1flow-throughfraction,4washfractions,and2elutionfractions.

9)Whentheinvitrotranslationreactionsarefinishedpassthereactionoverthespincolumninavolumeof100uL.

10)Setupthecolumnloadsample.50uLinvitroreaction(halfthesampleafterthespincolumn),130uLACbuffer,and45uL5%milkpowder.Totalvolume=225uL

11)Washthenickelresin.Aftertheproteinhasbeenallowedtocoupletotheresinfor30minutesonice,washtheresintwicein1mLofACbuffertoremoveunboundproteinandtoequilibratetheresininACbuffer.

12)Setupthecolumns.Add50uLofsettledaffinityresintothepreparedtips.Allowtheresintosettleonice.

13)Loadthecolumns.Aftertheresinhassettled(5-10minutes)removeanybufferremainingontop.Carefullyload90uLoftheloadsampletothetopofeachcolumn(controlandexperimental).DONOTDISTURBTHESETTLEDRESIN.TheremainingportionoftheloadsampleshouldbesavedforSDS-PAGE.

14)Allowtheloadedproteintoflowintotheresin.Whenitreachesthebottomofthecolumn(youcanfollowitsprogressbecauseoftheredcoloroftheretic.lysate)transferthecolumnintotube#1.Allowtheremainingproteintoflowintothetube.Thiscomprisesthemajorityoftheflow-throughfraction.Whenthelastoftheloadhasflowedintothecolumntransferthecolumntotube#2.

15)Washthecolumnswith4x400uLofACbuffer.Eachwashfractionis400uL.Transferthecolumntoanewtubeaftereachwashstep.

16)Elutetheboundprotein.Afterthelastwashtransferthecolumnstotube#6atroomtemperature.Add30uLof2%SDS(thiswouldprecipitateonice).Allowthistoflow-in.Transferthecolumntotube#7.Add120uLof2%SDS.Allowthistorunthrough.Thisistheeluatefraction.

17)Forgels:LoadandFlowThroughwillhave9uLsample+3ul4XSDSgelloADIngbuffer

Tocompensatefordilution,eluateloadedwillequal15ulsample+5uL4XSDS

18)Boilthesamples,storeinfreezeruntilreadyforSDS-PAGE.Afterelectrophoresis,drythegelandautoradiograph.Aninteractionisdetectedasdepletionofthelabeledproteinfromtheflow-throughandpresenceoftheproteinintheexperimentalcolumneluate.

AffinitychromatographywithFTZ(1st3lanes)andcontrol(2nd3lanes)affinitycolumns.35S-labeledPRDandFTZ-F1bindtotheFTZcolumnsandcanthenbeeluted(E).(L=load,FT=flowthrough;Luc=luciferase,negativecontrol).