ReagentsandMaterials

PBSPBS:0.05MEDTAPBS:0.01thimerosalConjugateStorageBufferSATADMFHydroxylaminehydrochlorideNaOHPelletsPD10ColumnorSephadexG25Sulfo-SMCCPreparationofThiolatedAntibody

Concentratetheantibodyto5-7mg/mlDissolve10mgofSATAin1mlDMF(SATA:DMF)Add3.0mlSATA:DMFpermgofantibodyGentlystirsolutionfor2hoursatroomtemperature.Dissolve0.5ghydroxylaminehydrochloridein10mlPBS:EDTAAdd0.25gNaOHpelletstothissolutiontoneutralizetoapproximatelypH7.0Addtoantibodysolutionat6.49ml/mlSATA.Gentlystirfor30minutesatroomtemperature.EquilibratecolumnwithPBS:EDTA.Desaltthiolatedantibodyoncolumn.CollectinthesmallestvolumepossIBLewithoutcollectinganySATA.AllowfreeSATA(2ndpeak)tocomeoffthecolumn.Storethethiolatedantibodyat2-8oCfornomorethanonehour.Re-equilibratethecolumnwithPBS.PreparationofMaleimide:HRP

Weighout1.067mgHRPforeachmgofthiolatedantibody.Add1.1mgSulfo-SMCCpermgofHRPAddPBStoafinalconcentrationof8mg/mlHRPMixfor2hoursonarotator.DesalttheHRPonPBSequilibratedcolumn.Collectthefirstpeak.PreparationofAb:HRPConjugate

ImmediatelymixtheHRPwiththethiolatedantibodyat1mgHRPpermgantibody.Gentlystirfor16-20hoursat2-8oC.Dialyzeagainst3changedofPBS,changingevery2hours,100Xtheconjugatevolume.Dilutetheconjugate2-foldinConjugateStorageBuffer.SterileFilter.