The antibody sticks to the protein. The enzyme produces a color by acting on a chromogen.
Chromogens turn color as a result of enzyme activity.
When the secondary antibody is present, BCIP acts as the substrate for alkaline phosphatase, and NBT enhances the navy-black final color of the precipitates at the reaction sites. The antibody hybridizations are carried out in a solution favoring specific binding. Do not allow the membranes to dry out during any of the following steps. Perform all of the subsequent washing and incubation steps at room temperature with gentle shaking. Float the membrane on TBST until it is evenly wet, submerge it and rinse briefly in the same buffer.