Peroxidase Conjugation by Periodate Method

Reagents

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

Procedure

1. Dissolve 16 mg HRP in 4 ml diH₂O and 0.8 ml of sodium periodate.
2. Stir for 20 minutes, RT
3. Add 6 drops ethylene glycol
4. Stir for 5 minutes, RT
5. Filter over a G-25 Sephadex column equilibrated with 1mM acetate
6. Pool colored fractions and divide into two equal parts.
7. To the first part, add 14 mg IgG
8. To the second part, add 10 mg IgG.
9. To both add 1M carbonate buffer, dropwise, to raise the pH to 9.0-9.5
10. Stir for 2 hours at room temperature
11. To both add 0.2 ml sodium tetraborate
12. Incubate for 2 hours at 2-8^oC
13.  
14. Dialyze against PBS.
15. Separate on ACA-44 column, eluted with PBS
16. Pool fractions and add 10 mg/ml PBS