Peroxidase Conjugation by Periodate Method

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