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        Peroxidase Conjugation by Periodate Method

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        897

         

        Reagents

        Horseradish peroxidase
        Sodium periodate, 0.1M, freshly made
        Ethylene glycol
        Sephadex G-25
        Sodium acetate buffer, 0.001M, pH 4.2
        IgG
        Carbonate:Bicarbonate Buffer, 1.0 M, pH 9.5
        Sodium tetraborate (4 mg/ml) freshly made.
         PBS
         Procedure

         Dissolve 16 mg HRP in 4 ml diH2O and 0.8 ml of sodium periodate.
        Stir for 20 minutes, RT
        Add 6 drops ethylene glycol
        Stir for 5 minutes, RT
         Filter over a G-25 Sephadex column equilibrated with 1mM acetate
        Pool colored fractions and divide into two equal parts.
        To the first part, add 14 mg IgG
        To the second part, add 10 mg IgG.
        To both add 1M carbonate buffer, dropwise, to raise the pH to 9.0-9.5
        Stir for 2 hours at room temperature
        To both add 0.2 ml sodium tetraborate
        Incubate for 2 hours at 2-8oC
        Dialyze against PBS.
         Separate on ACA-44 column, eluted with PBS
         Pool fractions and add 10 mg/ml PBS 

         

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