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同位素法测定底物磷酸化活性方法 Phosphorylation of Substrates
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PhosphorylationofSubstrates
ScottT.Eblen,N.VinayKumar,andMichaelJ.WeberDepartmentofMicroBIOLOGyandCancerCenter,UniversityofVirginiaHealthSystem,Charlottesville,VA22908ExcerptedfromProtein:ProteinInteractions,SecondEditionEditedbyEricaA.GolemisandPeterD.Adams
ABSTRACT
Ideally,onewouldliketobeabletodirectlyphosphorylatesubstratesinanintactcell.ThiscouldpotentiallybeperformedbyintroducingATPanalogintoliveorpermeABIlizedcells.However,cellsareimpermeabletoATP,andtheadditionoflabeledATPanalogtodigitonin-permeabilizedcellsresultsinthehydrolysisoftheanalogATPwithin1min(Chaudharyetal.2002).Therefore,wehavechosentoapplythistechniquetocelllysatesorfractions.Herewedescribetwoapproachestodetectdirectsubstratesofaproteinkinase.Thefirstapproachinvolvesidentificationofkinase-associatedsubstratesbyimmunoprecipitatingataggedformofthemutantkinasefromtransfectedCOS-1cellsandperformingakinasereactionbytheadditionof[γ-32P]ATPanalog.Thesecondapproachinvolvestheadditionofrecombinantmutantkinaseand[γ-32P]ATPanalogtocelllysates.WehaveusedthesetechniquesforthephosphorylationofERK2substrates;however,thismethodologycanbeappliedtootherproteinkinases.
MATERIALS(forApproachesIandII,asIndicated)
Buffers,Solutions,andReagents
  • Recombinantkinase(II)
  • [γ-32P]ATPanalog(I+II)
  • SepharoseCL-6Bbeads(I)
  • Lipofectamine(I)
  • AgoNIST(I),e.g.,epidermalgrowthfactor,serum,platelet-derivedgrowthfactor
  • FLAGM2agarosebeads(I)
  • SDS,20%(I)
  • M2lysisbuffer(II)(seeProtocol1)
  • PBS,roomtemperature(I)andicecold(I+II)(seeProtocol1)
  • Serum-freemedium(I,e.g.,Dulbecco"smodifiedEaglemedium)
  • FLAGpeptide(5mg/mlinhypotoniclysisbuffer)(I)
  • Kinasebuffercontainingbenzamidine,10mmand100mmNaCl(II)
  • 2xLaemmlisamplebuffer(I)
  • Hypotoniclysisbuffer(I)
  • 20mmHEPES(pH7.4)
  • 2mmEGTA
  • 2mmMgCl2
Cells
  • COS-1cells(I+II)
Plasmids
  • Maxi-prepplasmidDNAscarryingepitope-taggedversionsofthewild-typeandmutantformsofthekinaseofinterest(I)
SpecialEquipment
  • Tissueculturedishes(I+II)
  • Hypodermicneedle,11/4"",27gauge(I)
  • MiniprepColumns(I)
  • Dialysiscassette,10,000molecular-weightcutoff(II)
  • SDS-polyacrylamidegel(I)
  • Rotator,setupinacoldroom(4°C)(I)
  • Boilingwaterbath,presetto100°C(I)
  • Incubator,presetto37°C,5%CO2(I+II)
  • Incubator,presetto30°C(I+II)
  • Microfugecooledto4°C(I+II)
METHOD
ApproachI:PhosphorylationofKinase-associatedSubstratesItisimportanttostandardizetheprocedureinsmall-scalereactionspriortoscaling-uptheproceduretoidentifynovelsubstrates.
  1. Plate1x106COS-1cellsonto100-mmtissueculturedishes1daypriortotransfection.Incubatethecellsat37°Cin5%CO2.Transfectthecellswith6µgofplasmidDNAcarryingepitope-taggedversionsofthewild-typeormutantformsofthekinaseofinterest,and24µlofLipofectamine,accordingtothemanufacturer"sinstructions.
  2. 24-72hrposttransfection,washthecellstwicewithroom-temperaturePBSandserum-starvetheminserum-freemediumfor4-12hr.Thenstimulatethecellswithagonistfor5-10min.Dependingonwhichkinaseisbeingstudied,differentstarvationtimesandstimulantscanbeused.
  3. WashthecellstwicewithcoldPBSonice.Addhypotoniclysisbuffer(0.5ml/100-mmdish).HypotoniclysisbufferisutilizedtopreserveERK2interactionswithassociatedproteins.Foridentifyinginteractingsubstratesthatformstableassociations,M2lysisbuffer(seeProtocol1)oranotherbufferwithincreasingconcentrationsofdetergentorsaltscanbeused(HarlowandLane1999).
  4. Scrapethecellstogetherandtransferthemtoa1.5-mlmicrocentrifugetube.Donotvortex.Lysethecellsbycentrifugingat15,000ginamicrocentrifugefor20minat4°C.
  5. Transferthesupernatants(~1mgofprotein)intofreshtubes.Keepsmallaliquotsaside(20µg)tochecktheexpressionlevelsoftheexpressedproteins.
  6. WashtheappropriatequantityofSepharoseCL-6Bbeads(30µl/sample)withhypotonicbufferandmixwithFLAG-M2agarosebeads(10µl/sample),orbeadsconjugatedtoanotherantibodytothechosenepitopetag(~1µgofantibodyper1mgofproteinlysate).
  7. Washthemixtureofbeadstwiceinhypotoniclysisbufferandthenaddthemtothelysatespreparedinstep5(40µlpersample)forimmunoprecipitation.Incubatethebeadsinlysatefor1-2hrat4°Cwithconstantrotation.
  8. Washtheimmunoprecipitatesthreetimeswithhypotoniclysisbuffer(1mlperwashforeachsample).Afterthefinalwash,aspiratethefewremainingdropsofbufferusinga11/4inch,27gaugeneedle.
  9. Performkinasereactionsinatotalvolumeof40mlcontainingkinasebufferand10µCi[γ-32P]ATPanalogfor3-15minat30°C.Wefindthatshorterkinasereactiontimesarepreferable,butthetimeofincubationshouldbeoptimizedforeachkinaseandcelltype.Weusepilotreactionswithlabeledanalogtoidentifyconditionsforwhichthereactioncanbescaledup.Scale-upreactionsareperformedwithunlabeledATPanalogandtraceramountsoflabeledATPanalogontheimmunoprecipitate.Eventually,reactionsshouldbescaleduptogivesilverorCoomassiebluestainableamountsoftheproteinofinteresttoallowidentificationbymassspectrometry.
  10. Poollabeledandunlabeled(whenscalingup)kinasereactionsandadd80µlofFLAGpeptide(5mg/mlinhypotoniclysisbuffer).EluteFLAGERK2-QGanditsassociatedproteinsbyincubatingonicefor15minfollowedbyincubationat30°Cfor15min,withoccasionalvortexing.
  11. Resolvethesamplesin1xLaemmlisamplebufferona1-dimensionalSDS-polyacrylamidegel.
  12. ToperformisoelectricfocusingpriortoSDS-polyacrylamideelectrophoresis,add20%SDStothesamplestoafinalconcentrationof2%andboilthesamplesfor4min.RemoveSepharoseCL-6BandM2agarosebeadsbyloADIngthereactionintoaminiprepcolumnplacedinamicrocentrifugetubeandcentrifugingfor30secat15,000g.
ApproachII:PhosphorylationofSubstratesinCellLysateswithExogenousKinaseThisapproachusesrecombinantproteinkinasetophosphorylatesubstratesinacelllysate
  1. Platecellsandincubateat37°Cand5%CO2untilthecellsare80%confluent.WashthecellstwiceonicewithcoldPBS.Drainthedisheswell.Add0.5mloffreshM2lysisbufferper100-mmdishandscrapethecellsintomicrocentrifugetubesonice.Keepthesamplesonicefor15min,vortexingoccasionally.
  2. Centrifugethesamplesat15,000ginamicrocentrifugeat4°Cfor15min.Transferthesupernatanttoafreshtubeonice.
  3. Poolthelysatesanddialyzethemagainstkinasebuffer(withoutATPorDTT)containing10mmbenzamidineand100mmNaCl.Dialyzetwicefor2hreachin1literofbufferusinga10,000MWCODialysisCassette.ThiswillgetridofcellularATP.Performaproteinassayonthelysate.Dialyzedlysatescanbestoredat-70°Cuntiluse
  4. Addpurified,activerecombinantmutantkinaseto100µgofcelllysate.Weuse10-100ngofactivatedERK2,butthismustbedeterminedempiricallyaccordingtothespecificactivityofthekinaseofinterest.
  5. Add10µCiofradiolabeledATPanalog.Mixandincubateat30°Cfor3-15min.Wefindthatshortreactiontimes(3min)areoftenpreferable.Thisismostlikelyduetodepletionoftheanalogovertimeandtheabilityofphosphatasesinthereactiontodephosphorylatesubstrates.
REFERENCES
ChaudharyA.,BruggeJ.S.,andCooperJ.A.2002.Directphosphorylationoffocaladhesionkinasebyc-src:EvidenceusingamodifiednucleotidepocketkinaseandATPanalog.Biochem.Biophys.Res.Commun.294:293-300.HarlowE.H.andLaneD.L.1999.UsingAntibodies:ALaboratoryManual.ColdSpringHarborLaboratoryPress,ColdSpringHarbor,NewYork.
Anyoneusingtheproceduresinthisprotocoldoessoattheirownrisk.ColdSpringHarborLaboratorymakesnorepresentationsorwarrantieswithrespecttothematerialsetforthinthisprotocolandhasnoliabilityinconnectionwiththeuseofthesematerials.Materialsusedinthisprotocolmaybeconsideredhazardousandshouldbeusedwithcaution.Forafulllistingofcautionsregardingthesematerial,pleaseconsult:Protein:ProteinInteractions,SecondEdition:AMolecularCloningManual,editedbyEricaA.GolemisandPeterD.Adams,©2005byColdSpringHarborLaboratoryPress,ColdSpringHarbor,NewYork,p.457-461.
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