HIGH-RESOLUTION SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL-ELECTROPHORESIS AND IMMUNOCHEMICAL IDENTIFICATION OF THE 2X AND EMBRYONIC MYOSIN HEAVY-CHAINS IN COMPLEX-MIXTURES OF ISOMYOSINS

In mammals myosin heavy chains (MHC) are polypeptides with a molecular mass of about 200 kDa whose isoforms can be identified by sodium dode cyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoch emistry. Electrophoretic analysis is the only method for quantitating MHC profiles in single myofibers and/or cryostat sections of biopsied muscle. We present a method for SDS-PAGE of adult rat skeletal muscle which resolves MHC into four bands: 1, 2B, 2X, and 2A from the faster to the slower migrating band. Furthermore, embryonic MHC can be also r esolved in a complex mixture of isomyosins, e.g. developing or regener ating muscles. The method does not involve preparation of gradient gel s or electrophoresis at low temperature. Improved reproducibility is o btained by: (i) modification of the sample buffer; (ii) use of 7% poly acrylamide in the separating gel; (iii) control of pH of running buffe r by recirculation or change of the buffer during the run; and (iv) a 24 h run. The procedure is compatible with Coomassie Brilliant Blue, s ilver and immunoblot staining. Resolution is sufficient to permit tran sblotting of separated MHC after SDS-PAGE. The different isoforms are easily identified with monoclonal antibodies. The technique provides a n improved method to separate MHC and quantitate MHC2X and MHCemb in c omplex mixtures of MHC from a few cryostat sections of normal and dise ased muscle.