Urea‐glycerol‐acrylamide gel electrophoresis of acidic low molecular weight muscle proteins: Rapid determination of myosin light chain phosphorylation in myosin, actomyosin and whole muscle samples

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Abstract
We have improved the Perrie and Perry method (Biochem. J. 1970, 119, 31–38) of urea‐electrophoresis by the addition of a stacking gel containing 8.5 M urea and increased buffer capacity of the glycerol‐containing separation gel. The system has been successfully applied to the analysis of myosin phosphorylation levels not only in myosin and actomyosin, but also in whole muscle samples dissolved in urea. By subjecting the samples to a simple centrifugation and filtration step, all myosin light chains including the unphosphorylated and phosphorylated species as well as calmodulin and troponin could be identified. With a sensitivity of about 5 pmols the procedure described eliminates the need for the time‐consuming two‐dimensional electrophoresis which is commonly used to determine phosphorylation levels in muscle samples.