down jason walker

GST-PULLDOWN手册

冷泉港protocol——GSTpull-downProtocol

GSTPull-down

on,eva,ckThisprotocolwasadaptedfrom

"IdentificationofProtein-ProteinInteractionswithGlutathione-S-TransferaFusionProteins,"

Chapter6,inProtein-ProteinInteractions,2ndedition(sandAdams).ColdSpring

HarborLaboratoryPress,ColdSpringHarbor,NY,USA,2005.

INTRODUCTION

Glutathione-S-transfera(GST)fusionproteinshavehadawiderangeofapplicationssincetheir

heapplicationsis

thl-downmethod

desprecipitation

isbadontheabilityofanantibodytobindtoitsantigeninsolution,andthesubquent

purifirly,the

GSTpull-downisanaffinitycaptureofoneormoreproteins(eitherdefinedorunknown)in

solutionbyitsinteractionwiththeGSTfusionprobeproteinandsubquentisolationofthe

complexbycollectionoftheinteractingproteinsthroughthebindingofGSTto

glutathione-coupledbeads.

RELATEDINFORMATION

AnintroductiontotheuofGSTfusionproteinsforstudyingprotein-proteininteractionscanbe

foundinIdentificationofProtein-ProteinInteractionswithGlutathione-S-Transfera(GST)

FusionProteins.

Thisprotocolisdesig

35S-labelingprocedures,eOrlinickandChao(1996)andSpectoretal.(1998).Additionally,if

theinteractingproteinofinterestisknowntobeconfinedtoaspecificcellularcompartment(e.g.,

thenucleus),afractionofthecelllysatecorrespondingtothatcompartment(e.g.,anuclearextract

[toprepare,eDignametal.1982])canbeudinplaceofatotalcelllysate.

MATERIALS

ThisproceduremayrequireequipmentorreagentsforWesternanalysis,Coomassiebluestaining,

and/orsilverstaining(eStep13).

Reagents

Celllysate(unlabeledorlabeledwith35S,dependingonexperimentalgoal)

ThixperimentcomparesGSTversusGSTfusionprotein,soitisnecessarytoprepareenough

untoflysateneededtodetect

ithlysateequivalentto1x106to1x107tissueculture

cells.

GSTfusionprotein(ePreparationofGSTFusionProteins)

GSTprotein

GSTpull-downlysisbuffer,icecold

ReagentsforSDS-PolyacrylamideGelElectrophoresisofProteins(eStep12)

2XSDSgel-loadingbuffer

Tris-Cl(50mM,pH8.0)containing20mMreducedglutathione(optional;forStep11only)

GST-PULLDOWN手册

Equipment

EquipmentforSDS-PolyacrylamideGelElectrophoresisofProteins(eStep12)

Geldryer(optional;eStep13)

Glutathione-Sepharobeads(storeat4°C;donotfreeze)

Beadsaportant

towashthebeadsthoroughlyinGSTpull-downlysisbufferandtogeneratea50/50slurryof

beadsinGSTpull-downlysisbufferpriortou.

Microcentrifuge,precooledto4°C

Microcentrifugetubes,0.5mL(optional;)and1.5mL

Needle,smallbore,sterile(optional;)

Rotatorforend-over-endmixing

Waterbath,boiling(optional;eStep10)

X-rayfilm(optional;eStep13)

METHOD

tethecelllysatewith50μLofglutathione-Sepharobeads(50/50slurryinlysisbuffer)

and25μgofGST(NOTtheGSTfusionprobeprotein)for2hat4°Cwithend-over-endmixing.

Allowenoughvolumeinthetubetopermitliberalmixing;500μlto1mLisagoodstarting

point.

Thisstepisdesignedtopreclearfromthelysateproteinsthatinteractnonspecificallywiththe

nteractionwillbedetectedprimarilywithantibodies

directedtoacandidateinteractingprotein,itisnotabsolutelynecessarytopreclearthelysates

r,when35S-labeledcelllysatesareudto

identifynovelprotein-proteininteractions,thestepscanhelptoreducebackground.

Whendetectingtheinteractingproteinwithantibodiestothatprotein,itisimportanttoinclude

"GST+beads"and"beadsalone"controls.

fugeat13,000rpmfor10cat4°Cinamicrocentrifuge.

erthesupernatant(preclearedcelllysate)toafreshtube.

wotubescontainingequalamountsofthepreclearedcelllysate.

50μlofglutathione-Sepharobeads(50/50slurryinlysisbuffer)toeachtube.

dGSTproteintoonetubeandtheGSTfusionprobetotheother(~5-10μgeach).

Theamountofproteinaddedshouldbeequimolarinthetworeactions(i.e.,thefinalmolar

concentrationofGSTshouldbethesameasthatoftheGSTprobeprotein).

tethetubesfor2hat4°Cwithend-over-endmixing.

fugethesamplesat13,000rpmfor10cat4°Cinamicrocentrifuge.

erthesupernatantstofreshmicrocentrifugetubesandrervethemforSDS-PAGE(e

Troubleshooting).

GST-PULLDOWN手册

dthewashes.

point,herthe

boilingmethod(themorepopularchoice;eStep10)oroneoftheelutionmethodsdescribedin

Step11.

Thedisadvantageofelution(Step11)isthatifmultipleelutionsarenecessary,thefinalsample

ecas,itmaybeimpossibletoloadmorethanasmallpercentageof

thereasonmostrearcherschoo

insteadtoboilcomplexesoffthebeadsinSDSsamplebuffer(Step10).

amplesaretobeboiledoffthebeads,doasfollows:

qualvolumeof2XSDSgel-loadingbuffertothebeads.

Itisimportanttoincludea"glutathione-Sepharobeadsonly"nsbound

nonspecificallytothebeadscanappearasboundtothefusionprotein,evenincomparisontoGST

alone.

r5mininawaterbath.

SamplesarenowreadyforSDS-PAGE(Step12).

amplesaretobeelutedfromthebeads,performoneofthefollowingoptions:

OptionI:

50μlof20mMreducedglutathionein50mMTris-Cl(pH8.0).

hetubetomixthecontents,andincubatethesamplesatroomtemperaturefor5min.

fugethesamplesat13,000rpmfor10catroomtemperatureinamicrocentrifuge.

erthesupernatantcontainingtheelutedproteintoanewmicrocentrifugetube.

ionisincomplete,.

OptionII:

50μlof20mMreducedglutathionein50mMTris-Cl(pH8.0).

hetubetomixthecontents,andincubatethesamplesatroomtemperaturefor5min.

lly,usingasterile,small-boreneedle,poke

tinsidea1.5-mLmicrocentrifugetube.

fugethetubestogetherat1000ginamicrocentrifugefor2minatroomtemperature.

Theelutedproteinswillbedepositedinthe1.5-mLtube.

easmuchofthesampleaspossiblebySDS-PAGE(eSDS-PolyacrylamideGel

ElectrophoresisofProteins).

hodofdetectionwilldependontheexperimentalgoal:

oalistodetectthe35S-labeledproteinsassociatedwiththefusionproteinafter

SDS-PAGE,drythegelonageldryerandexpoittoX-rayfilm.

oalistodetectspecificpartnersafterSDS-PAGE,transfertheproteinstoamembrane

andsubjectthemtoWesternanalysis.

GST-PULLDOWN手册

oalistodeterminethesizeandabundanceofproteinsassociatedwiththefusionprotein

fromanonradioactivelysate,subquenttoSDS-PAGE,stainthegelwithCoomassieblueor

silverstain.

TROUBLESHOOTING

Problem:Conditionsforpull-downarenotoptimal

[Step13]

Solution:Analyzealiquotsofequalpercentagevolumes(e.g.,1%)fromeachofthefollowing

fractionsgeneratedduringtheprotocol:

Totalcelllysate(Step1)

Beadspriortoelution(Step9)

Eluate(Steps10or11)

Beadspost-elution(Steps10or11)

SupernatantsavedatStep7

"Beads+GST"eluate(ifapplicable;Steps10or11)

"Beadsalone"eluate(ifapplicable;Steps10or11)

Afterthesamplesarecollectedattheindicatedsteps,addanappropriatevolumeofSDS-PAGE

-freezethesamplesinadry-iceethanolbathforanalysisbySDS-PAGE

(duringStep12),andperformautoradiography,Westernanalysis,orCoomassiestainingas

appropriate(Step13).

Resultsfromthisgelwillshow:

Theprevalenceofthenovelinteractorinthetotalcelllysate(aliquotfromStep1).

HowmuchoftheinteractorisboundtotheGSTfusionprotein(aliquotfromStep9).

HowmuchGSTfusionprotein+associatedproteinswereelutedfromthebeads(aliquotofeluate

fromSteps10or11).

Whatfractionoftheinteractorsremainedbound(beadsremainingfromSteps10or11).

Howmuchoftheinteractingproteinwasdepletedfromthetotalcelllysate(aliquotfrom

supernatantatStep7).

Problem:Lowsignal

[Step13]

Solution:Ifaninteractionyieldsalowsignaleventhoughtheinteractorisabundantinthetotal

celllysate,einsaltand

detergentconcentrations,inadditiontoincreasingthetimeallowedforassociation,mayimprove

ignalcanalsobecaudbyinefficientelution;i.e.,thecomplexbeing

nbedeterminedbySDS-PAGEcomparison

oftheeluateversusthe"beadspost-elution"fraction(Steps10or11).Ifthisprovestobethe

problem,itmayberemediedbypoolingmultipleelutionsorincreasingthetimeforelution.

Releasingtheinteractorsbyboilinginsamplebuffer(Step10),ifappropriate,wouldbeapplicable

inthisca.

GST-PULLDOWN手册

Problem:Nonspecificbackground

[Step13]

Solution:PreclearingalysatewithGST,orbeadsalone,canhelptominimizenonspecific

interactions(eStep1).Titratingtheamountoflysateaddedandincreasingthestringencyofthe

bindingandwashconditionscanalsoreducebackground.

DISCUSSION

WhenanalyzinganinteractionbyWesternblot(e.g.,analyzingapredictedinteractionforwhich

thereareavailableantibodies),itisimportanttoreprobethemembranewithanti-GSTantibodies

lldeterminewhetherallsampleswereincubated

withthesameamountofGSTfusionprotein,anditwillhelptodeterminewhetherthefusion

nteractorsareradio-

orbiotin-labeled,oneshouldalsoconfirmequalamountsofGSTfusionandGSTproteinsby

WesternblotorCoomassiebluestaining.

REFERENCES

Dignam,J.D.,Lebowitz,R.M.,andRoeder,tetranscriptioninitiationbyRNA

cAcidsRes.11:

1475–1489.

Orlinick,o,ctionsofthecellularpolypeptideswiththecytoplasmic

.271:8627–8632.[Abstract/FreeFullText]

Spector,D.L.,Goldman,R.D.,andLeinwand,:ring

HarborLaboratoryPress,ColdSpringHarbor,NY.