High-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical identification of the 2X and embryonic myosin heavy chains in complex mixtures of isomyosins

K. Rossini, C. Rizzi, M. Sandri, A. Bruson, U. Carraro

Research output: Contribution to journalArticle

Abstract

In mammals myosin heavy chains (MHC) are polypeptides with a molecular mass of about 200 kDa whose isoforms can be identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunochemistry. 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 resolved in a complex mixture of isomyosins, e.g. developing or regenerating muscles. The method does not involve preparation of gradient gels or electrophoresis at low temperature. Improved reproducibility is obtained by: (i) modification of the sample buffer; (ii) use of 7% polyacrylamide in the separating gel; (iii) control of pH of running buffer by recirculation or change of the buffer during the run; and (iv) a 24 h run. The procedure is compatible with Coomassie Brilliant Blue, silver and immunoblot staining. Resolution is sufficient to permit transblotting of separated MHC after SDS-PAGE. The different isoforms are easily identified with monoclonal antibodies. The technique provides an improved method to separate MHC and quantitate MHC2X and MHCemb in complex mixtures of MHC from a few cryostat sections of normal and diseased muscle.

Original languageEnglish
Pages (from-to)101-104
Number of pages4
JournalElectrophoresis
Volume16
Issue number1
DOIs
Publication statusPublished - 1995

Keywords

  • isomyosins
  • muscle plasticity
  • myosin heavy chains
  • rat muscle
  • sodium dodecyl sulfate-polyacrylamide gel electrophoresis

ASJC Scopus subject areas

  • Clinical Biochemistry

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