Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Protein Staining on Nitrocellulose Paper
Detection Using Antibodies

Proteins transferred to nitrocellulose membranes are treated with 2,4-dinitrofluorobenzene. The resulting protein derivatives can be detected at levels as low as 10 ng using Antibodies against dinitrophenyl (DNP) groups via the peroxidase-antiperoxidase complex method. This approach is 100 times more sensitive than Coomassie staining while avoiding the complications associated with silver staining.

Reagents.

A. 50 mM NaHCO3 in 50% dimethyl sulfoxide.

B. 0.14 M NaCl buffered with 10 mM Na-phosphate, pH 7.2.

C. 3% BSA—10% heat-inactivated calf serum in 0.14 M NaCl buffered with 10 mM Na-phosphate, pH 7.2.

D. 50 mM Tris-HCl buffer (pH 7.5) containing 0.3 mg/ml 3,3'-diaminobenzidine and 0.005% H2O2.

Procedure.

1. Incubate the nitrocellulose membrane for 10 min at room Temperature in Reagent A containing 0.00001–0.1% 2,4-dinitrofluorobenzene. Longer incubation times and higher reagent concentrations may complicate the removal of excess reagent. Wash several times with Reagent A. Antigenic determinants are identified according to [368], as described below.

2. Wash the blot with Reagent B (five times for 5 min each).

3. Incubate with gentle agitation for 1 h at 40 °C in Reagent C.

4. Incubate overnight in rabbit antiserum against DNP groups diluted (1:200) with Reagent C.

5. Wash the membrane with Reagent B (4 times for 5 min each).

6. Incubate for 30 min at room temperature with goat antibodies against rabbit IgG (Serasource) diluted (1:40) with Reagent C.

7. Wash with Reagent B (4 times for 5 min each).

8. Incubate for 30 min at room temperature with the peroxidase-antiperoxidase complex (Miles) diluted (1:40) with Reagent C.

9. Wash with Reagent B (3 times for 5 min each).

10. Immerse in Reagent D to detect peroxidase activity.



Last update: 06/08/2026

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