ISOLATION OF UNSTABLE MYOSINS AND THE ANALYSIS OF LIGHT-CHAINS BY CAPILLARY ELECTROPHORESIS

A rapid method for the isolation of unstable fish myosins by Sepharose Q ion-exchange chromatography is described which yields a pure protein essentially free of contamination or breakdown products in less than 6 h. A protocol was developed for determining the molar ratios of myosin light chains (LC) by capillary electrophoresis. The method is quantitative, rapid (<20 min), highly reproducible (<2.1% variation in relative peak migration time), and only uses Femtomole quantities of protein. It was able to separate myosin Light chains which could previously only be resolved by 2-D sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Capillary electrophoresis in the presence of SDS gave similar apparent relative molecular masses (M(r)) for most proteins, but an anomalously high M(r) for myosin light chain 3 (LC3), as has been reported previously for SDS-PAGE methods. (C) 1995 Academic Press, Inc.