A variation of ECL immunodetection method permits sequential detection of multiple antigens (MAD) on a single protein blot without stripping previously bound antibodies. Because antibody stripping is not involved, immobilized proteins are not lost from the membrane, which permits multiple sequential reprobings of the same membrane with different primary antibodies (> or = 12) and retention of strong signal intensities for all antibody probings. This procedure utilizes horseradish peroxidase (HRPase)-based detection with both chemiluminescent and colorimetric substrates. Initial incubation of the blot with secondary antibody followed by colorimetric development prior to probing the blot with primary antibodies markedly reduces background intensities in ECL-based detection procedures and permits sequential use of antibodies derived from a single species. By allowing large amounts of data to be obtained from a single blot, MAD immunoblotting has the potential to markedly streamline the work required to compare the expression levels of several proteins within biological samples. This technique could be particularly valuable for analyzing cellular populations that are difficult to isolate in large numbers or clinical specimens where the amount of protein samples is limited or available on a one-time basis.