MaterialsForprocedure1•0.1Mphosphatebuffer,pH7.0•Tris-bufferedsaline(TBS)•5%BSAinTBS•stabilizersoln(10%sodiumthiosulfate+0.1NNaOH)•IODO-BEAD™(Pierce)•Hamiltonmicrosyringe(model702,25µl,needlegauge22S,pointstyle#2)•desaltingcolumn(Bio-RadDG-10etc.)•columnstandForprocedure2•PBScontaining20mMglucose•Hepes-Tyrodebuffer(oranybuffercompatIBLewithyoursubsequentexperimentswithlabeledcells)•lactoperoxidase(SigmaL-8257)dissolvedinPBSat1mg/ml•glucoseoxidase(SigmaG-7016)dissolvedinPBSat5U/mlProcedurei)Radioiodinationofproteininsolution*ReadingafreebookletavailablefromAmersham("Guidetoradioiodinationtechniques")isrecommended.1.Inclearplastictube,add200µlof0.1Mphosphatebuffer,pH72.Add5µl(500µCi)of125-Iusingmicrosyringe3.Add1-2piecesofIODO-BEADs¶4.incubateatroomtemp,5min5.Addyourprotein(1-100µg,25µgisgoodforIgGsorFab)6.Incubateatroomtemp,10-25min7.Takereactionmixture(leavebeads)andapplyonadesaltingcolumn§8.Collectfractions(0.5-1ml/fr.)¶Priortouse,washedthreetimeswithphosphatebuffertoremovedebrisonthebeads)§Blocknonspecificbindingsitebyapplying1mlof5%BSA-TBSfollowedbyequilibrationwithTBS. 
TypicalresultwithFabfragmentlabeling(50µgused) Labelingefficiency(incorporation)shouldbe10-90%(dependsonamountofproteinadded).Collectappropriatefraction(inthiscase,fr#3+4),add1/4volof5%BSA*,andstoreeitherat4°Cor-80°Cinaliquots.*additionofhighconc.BSAwillguardyourdiluteproteinfromdecompositionbyradiation,butshouldnotaddedifitinterferewithsubsequentexperiments.However,labeledanddiluteproteinwithoutcarrierproteintendstoflocculateandeasilylosesitsactivity.ii).Radioiodinationofcellsurfaceproteins1.WashcellsatleastthreetimeswithPBSandresUSPendin1mlofPBScntg.glucoseat0.5-2x107cells/ml)in15mlconicaltube,holdonice2.Add5-50µl(0.5to5mCi)125Iusingmicrosyringe3.Add50µloflactoperoxidase(1mg/ml)4.Add10µlofglucoseoxidase(5U/ml)5.onice,5min(occasionallyagitate)6.Addadditional10µlofglucoseoxidase(5U/ml)7.onice,5min(occasionallyagitate)8.Addadditional10µlofglucoseoxidase(5U/ml)9.onice,5min(occasionallyagitate)10.Add6mlofPBS(termination)11.spindownthecells(1500rpm,3min)**transferthesupernatanttowastebottlecontainingstabilizingsolution12.Wash2moretimeswithPBS13.Suspendthecellsinappropriatebufferforsolubilizationofcellsurfaceproteins(successfulexperimentswillresultin10-30%incorporationof125Iintocells)References1."Guidetoradioiodinationtechniques"freebookletavailablefromAmersham2.Marchalonis,Biochem.J.,113,299(1969)3.Krameretal,CancerRes.,49,393(1989)
