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