Phosphorylation analysis of contractile regulatory proteins in smooth muscle by using phos-tag SDS electrophoresis

Abstract

Activation of myosin by phosphorylation has been implicated in regulation of smooth muscle contractions. The ability to analyze myosin phosphorylation is crucial for better understanding of the regulatory mechanisms of smooth muscle contraction. In myosin phosphorylation analysis, it is important to quantify the ratio between phosphorylated and unphosphorylated myosin because those can have opposite effects on contractions. For this reason, urea/glycerol-PAGE and 2-D electrophoresis in combination with Coomassie blue staining or Western blotting are commonly used to separate and quantify myosin based on its phosphorylated state. However, those conventional techniques are not sensitive enough to analyze minute smooth muscles, such as terminal resistant arterioles, partly because of a poor separation in electrophoresis with a small sample. Recently we overcame this problem by introducing a phos-tag technique, and achieved picogram sensitivity. In this article, the sensitive phosphorylation quantification method and application of phos-tag electrophoresis to smooth muscle physiology research are reviewed.