PurposeandbackgroundsAboutnuclide...35-SDecay:(b-ray,0.167MeV)Halflife:87.5days
- Thickacrylicshieldcanblockmostoftheemission.Becauseofthelowenergy,itisimpossIBLetodetectspillage/contaminationbyusualsurveymeter.Wipetest+LSCisnecessary.
- Chemicalproperties...Useeither35S-methionineorcysteineorboth.RecommendusingExpre35S35SProteinlabelingmix(NEN).Beforeopeningthecapofthevial,thawthesolutioncompletelyandopenthecapinthehood(athotlab)sothatanyvaporizedmaterialscanbetrappedinthehood.
- PrincipleofrADIolabelingCellsincorporate35S-methionineorcysteineduringtheproteinsynthesis.ThusitisessentialtouseMet,Cys-freemediumanddialyzedFCSduringthelabeling.Shortperiodofincubationwith35S-methionineorcysteinewillresultinradiolabeling(pulse),andadditionalincubationwithexcessconcentrationofunlabeledMet+Cys(chase)isneededforcomplexglycoproteinslikeintegrinstogetexpressedasamaturatedform.Typically,overnightchaseisrecommendedformetaboliclabelingoffullyprocessedformofintegrins,butoptimumtimemayvarydependingoneachprotein.
- Materials
- •Completemedium:DMEMsupplmentedw/10%FCS,NEAA,Na-pyruvate,andGln•Expre35S35SProteinlabelingmix(NEN,#NEG-072),7.9mCi/ml•Transfectionreagents:CaCl2,x2HBS,DNA•LabelingMedium:RPMI1640(Met,Cys-free;SigmaR-7513)supplementedw/10%dialyzedFCS,&Gln•ChaseMedium:Labelingmedium(above)+500µg/mlCysteine-HCl,100µg/mlmethionine,sterilefiltered•Tris-bufferedsaline(TBS):20mMTris,150mMNaCl,pH7.4•X1000Proteaseinhibitorsoln:500mMPMSF(SigmaP-7626)inEtOH,10mg/mlleupeptin(SigmaL-2023)inwater,10mg/mlpepstatinA(P-4265)indimethylformamide•IPbuffer:1%Tritonx-100,0.05%NP-40inTBS•x2IPbuffer:2%Tritonx-100,0.1%NP-40inTBS•ProteinGagarose(Gibco#15920-010)•Gelfixationsolution:7%aceticacid,25%MeOHinwater
- •Gelsoakingsolution:1%glycerol,5%PEG8000inwater
- Procedure
- Transfectionof293cells(refertotheprotocol#4,"TransientTransfectioninto293TCells")Preparetransfectedcellsin6-wellplate.Use2µgDNA/subunit/well.After7-9hincubationwithDNA:calciumphosphate,rinseonceandreplacewithfreshmedium(3ml/well).Metaboliclabeling1.Transfectedcells->24haftermediumchange,rinsecellstwicewithLabelingMedium.2.Add1.5ml/wellLabelingMedium3.Add20-30µl(~250µCi)of[35S]methionine/cysteine/well4.Culture1hat37°C(pulse)5.Add1.5ml/wellChaseMedium6.Cultureo/n(chase)7.Harvestmetabolicallylabeledcells(andsupernatant)Immunoprecipitationi)preparationofcelllysate(formembraneorcytosolicproteins)1.Afterremovingthemedium,detachcellsbysUSPendinginTBS,transfertomicrofugetube2.Spindown&resuspendin0.5mlTBS3.Add1µleachofx1000proteaseinhibitorsoln4.Solubilizecellsbyadding0.5mlx2IPbuffer5.onicefor20min6.Centrifugeatmaximumspeedfor15min7.Savesupernatant(35S-labeledcellextract)ii)preparationofculturesupernatant(forsecretedproteins)1.Harvestmedium(into15mltube)2.Spindowntoremovecelldebries3.AdjustpHbyadding50µl1MTrispH8.04.Savesupernatant(35S-labeledculturesup)
- iii)IP1.Prepare1.7mlEppendorftubes(makesurethatcapclosestightly)2.Add100-400µlof35S-labeledculturesuporcelllysate3.AddmAb(1µgforpurifiedIgG,1µlforascites,or50µlforhybridomasupernatant)4.Add20µlofProteinG-agarose(50%slurrry)>>>usewide-holetipsfortransferringgelsuspension
- 5.Adjusttotalvolto500µlwithTBS(forsecretedproteins)orIPbuffer(forcellularproteins)6.Incubateat4°Cfor1-3h(Userocking-typemixerratherthanhorizontalshaker.Thesuspensionmustbemixedwellduringtheincubation.USESPECIALCAUTIONTOAVOIDRADIOACTIVESPILLAGE.Alwaysplaceprotectivesheets.)7.Spindown@6000rpmfor3min(usecentrifugeallowedforradioactivestuff)8.Removesupernatant(Leave~30µlofsolutionabovethepellet.Don’ttrytoremoveALLofthesolutionbecauseyouwillloseyouragarosebydoingso.)9.Resuspendin500µlofTBS(orIPbuffer)andspindown
- (repeatthiswashingstep3times)10.RemovefinalwashsolutionroughlywithP1000pipet,thencompletelywithnarrow-mouth,gel-loadingtipattachedtoP200pipetsothatyouwon’ttakeoutProteinGbeads.11.Totheslightlymoistpelletofbeads,add30µlSDS-PAGEsamplebuffer(eitherreducingornonreducing,dependingonthepurpose)12.Waitfor5min,thenboilfor1min13.Spindown,andload5-10µlontoSDS-PAGEgel14.Afterelectrophoresis,fixgelinfixationsoln.for30min15.Removesolutionandputgelsintoenhancersolution(EN3HANCE;NEN)for1h16.TransferEN3HANCEsolutiontowastebottle,soakgelsintosoakingsolution(30min)17.Drygels18.Autoradiography(useintensifyingscreen)KeywordsTransfection,293cells,35-S,metaboliclabeling,immunoprecipitation,autoradiography,monoclonalantibody
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