Dear all,

I want to label IgG with HRP, the following protocol have to seperate labelled antibody with colum, which may cause sample lose. Does anyone have procols avoiding colum seperation steps without commercial kits.

Thanks!

1.Dissolve 4 mg of peroxidase in 1 mL of water.

2. Add 200 µL of freshly prepared 0.1 M sodium periodate, and stir the solution for 20 min at room temperature.

3. Dialyze the modified enzyme against 1 mM sodium acetate buffer (pH 4.4) overnight

at 4°C or exchange the buffer using 30.000 MWCO centrifugal filter devices  (Amicon Ultra, Millipore) and resuspend modified enzyme in 1 ml of 1 mM sodium acetate buffer (pH 4.4)

5. Add 1 ml of IgG solution resuspended in  0.01 M sodium carbonate buffer (pH 9.5) (IgG concentration 8 mg/ml) and adjust the pH of the solution by adding 0.2 M sodium carbonate buffer (pH 9.5) to obtain pH 9.5. Stir the mixture for 2 h at room temperature.

6. Add 100 µL of freshly prepared 0.1 M sodium borohydride solution, and stir the

mixture at 4°C overnight. (*)

7. Dialyze the conjugate against PBS overnight at 4°C or exchange the buffer using 100.000 MWCO centrifugal filter devices  (Amicon Ultra, Millipore) and resuspend the conjugate in 1 ml of PBS. Add 50% Glycerol, 1% bovine serum albumin and 0.02% Thimerosal. Store at –20 °C.

(*) To remove conjugate aggregates, fractionate the mixture obtained at paragraph 6 by gel filtration in PBS and determine the A280 and A403.

Pool the fractions in the first peak (both A280 and A403 peaks coincide).