Protocols for Myosin and Actin-Myosin Assays Using Rapid, Stopped-Flow Kinetics

Fast transient kinetics using stopped-flow fluorimetry is now a powerful method for defining the ATPaseATPase cycle of myosinMyosins and its subfragments and has found wide use in defining the difference between myosinMyosins isoforms, myosinsMyosins carrying disease linked mutations, and the effect of small molecules on the ATPaseATPase cycle. Here the protocols for completing the classical assays of myosinMyosins and actin.myosinMyosins using the stopped-flow are described. The assays include ATPATP and ADPADP binding to myosinMyosins and actin.myosinMyosins, displacement of ADPADP from myosinMyosins and actin.myosinMyosins, and the cleavage of ATP to ADPADP and phosphate on myosinMyosins. Single and multiple turnover assays are also described.