A multiplex system of Western blotting is presented in which most of the current muscular dystrophy proteins can be analyzed simultaneously on one pair of blots. This represents a significant improvement in efficiency and cost for this type of analysis. The final diagnosis is more quickly achieved in patients where several possible diagnoses are indicated after clinical appraisal, and those with unusual presentations may be quickly resolved. The method uses a biphasic polyacrylamide gel system, which enables the corresponding blot to be probed simultaneously with a cocktail of monoclonal antibodies. The gel is optimized so that large proteins of more than 200 kd (eg, dystrophin, dysferlin, and myosin heavy chain) can be analyzed in the top part, while smaller proteins under 150 kd (eg, calpain 3, the 80-kd fragment of laminin alpha2 chain, all of the sarcoglycans, and caveolin 3) are separated in the lower phase. This basic system could be used for different combinations of antibodies as new muscular dystrophy proteins are identified and require examination. In addition, analysis of the laminin alpha2 chain of merosin showed that this protein was expressed as a doublet or triplet set of bands in many patients with active muscle pathology. This may indicate the existence of an embryonic isoform, which is re-expressed in regenerating fibers.