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Updated: Aug 10, 2026

Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays
Published on: February 4, 2021
Biochemical studies of myosin
1Department of Molecular Physiology and Biophysics, Given E205, University of Vermont, Burlington, Vermont 05405, USA. trybus@salus.med.uvm.edu
Abstract:
This article describes methods for expressing and obtaining purified smooth muscle myosin subfragments using the baculovirus/insect cell expression system, as well as methods for purifying whole myosin from tissue. Protocols for several gel assays that are routinely used with myosin are given, including gels to monitor light chain phosphorylation state and native gels to determine protein homogeneity. Steady-state myosin ATPase and actin-activated ATPase determinations are described, as are some of the more basic transient-state kinetic parameters that can be measured. The in vitro motility assay, in which the rate of actin movement over myosin or its subfragments is quantified, is also presented.
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Myosin II is a hexamer comprising two heavy chains with globular heads and coiled-coil tails, two regulatory light chains, and two essential light chains. The ATPase sites on the myosin heads hydrolyze ATP, and the released phosphate generates the force for contraction. It is...

