Chapter 16 Biochemical and Immunocytological Characterization of Intermediate Filaments in Muscle Cells

Abstract

Publisher Summary This chapter describes the biochemical and immunocytological characterization of intermediate filaments in muscles cells. An indispensable tool for the unambiguous characterization of the subunits of intermediate filaments in both muscle and nonmuscle cells has been the technique of two-dimensional isoelectric focusing (IEF)/sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The comparative one-dimensional peptide mapping of total smooth- and skeletal-muscle desmin has revealed the presence of peptide differences in desmin within a given species and among species, indicating that although desmin is a conserved molecule, it may be coded by different genes in the different muscle types and species. The two desmin variants co-purify with desmin purified from chicken gizzard through a number of cycles of polymerization–depolymerization, indicating that they are both integral components of desmin filaments and of the desmin molecule. It is found that proteolysis of vimentin can be avoided if the cells are first cross-linked with the reversible cross-linker dimethyl-3,3-dithiobispropionimidate, then lysed with 0.5% NP-40, and finally denatured and reduced with 9.2 M urea-0.5% P-mercaptoethanol prior to IEF.