Chapter 17 Molecular Diversity of Myofibrillar Proteins: Isoforms Analysis at the Protein and mRNA Level

Abstract

Publisher Summary This chapter describes and discusses the techniques used to identify the various isoforms of contractile proteins present in skeletal and cardiac muscle. The approaches presented, although mainly focus on contractile proteins, may also apply to isoforms of sarcotubular system proteins and of other muscle-cell components. Isoforms can be distinguished and their relative abundance can be evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional electrophoresis. These techniques must be integrated with immunoblotting analyses and/or with microsequencing and mass profile fingerprinting to establish the identity of electrophoretic bands and spots. The differential distribution of the isoforms among the various fiber types or the coexistence of two or more isoforms within the same fiber can be analyzed in tissue sections by immunohistochemical methods or in isolated single fibers by biochemical micromethods. The functional role of contractile protein isoforms can be explored by correlated biochemical and physiological studies of skinned single fibers. Isoform analysis at the mRNA level is essential for identifying new isoforms and for defining the mechanisms of gene regulation implicated in differential isoform expression.