hrp labeling of antibodies - (Feb/01/2007 )
can anybody help me how to label the antibodies with hrp enzyme
thanx in advance
if i am not wrong.. there are several methods u can use.. glutaraldehyde coupling is one of them i think.. not too sure abt the names.. but i am sure if u do a search online for coupling methods.. u can easily find them..esp for hrp to ab
Here is a protocol for labeling
1.HRP, 1000 U/mg or greater (with 2,2'-azino-bis-[3-ethylbenzthiazoline-6-sulfonic acid]
2. IgG antibody. This should be the pure IgG fraction or, better, affinity purified antibody
from an antiserum or pure monoclonal antibody.
3. 0.1 M Sodium periodate.
4. 1 mM Sodium acetate buffer, pH 4.4.
5. 10 mM Sodium carbonate buffer, pH 9.5.
6. 0.2 M Sodium carbonate buffer, pH 9.5.
7. Sodium borohydride, 4 mg/mL (freshly prepared).
8. Sepharose CL-6B (Amersham Pharmacia Biotech, Uppsala, Sweden) or a similar
gel filtration medium.
9. Phosphate-buffered saline (PBS): 20 mM sodium phosphate buffer, pH 7.2, containing
0.15 M NaCl.
10. Bovine serum albumin (BSA).
1. Dissolve 2 mg of peroxidase in 500 µL of water.
2. Add 100 µL of freshly prepared 0.1 M sodium periodate, and stir the solution for 20 min at
room temperature. (The color changes from orange to green.)
3. Dialyze the modified enzyme against 1 mM sodium acetate buffer, pH 4.4 (2 L) overnight
at 4 C.
4. Dissolve 4 mg of IgG in 500 µL of 10 mM sodium carbonate buffer, pH 9.5.
5. Adjust the pH of the dialyzed enzyme solution to 9.0–9.5 by adding 10 µL of 0.2 M sodium
carbonate buffer, pH 9.5, and immediately add the IgG solution. Stir the mixture for 2 h at
6. Add 50 µL of freshly prepared sodium borohydride solution (4 mg/mL), and stir the
mixture occasionally over a period of 2 h at 4ｰC.
7. Fractionate the mixture by gel filtration on a column (approx 1.5 . 85 cm) of Sepharose
CL-6B in PBS. Determine the A280 and A403.
8. Pool the fractions in the first peak (both A280 and A403 peaks coincide), add BSA to give
a final concentration of 5 mg/mL, and store the conjugate in aliquots at –20 C.
Glad to see you on another orbit!