All steps are conducted at room temperature and with gentle agitation on a rocking agitator.
1. Block nonspecific binding sites in the membrane with three washing steps, each 10 min in duration, in blocking solution (see Notes 12-14).
2. Incubate overnight in the primary antibody solution at the suitable dilution (see Note 15) in blocking solution.
3. Wash 3 x 10 min in blocking solution.
4. Incubate for 2 h in the secondary antibody solution (see Note 16).
5 Wash 3 x 10 min in blocking solution.
6. Wash 30 min in washing solution.
After this step one can go forward with ECL detection (see Notes 17 and 18). Alternatively, one can choose detection with the chro-mogenic substrate (see Note 19).
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