Product Uses
- Measurement of F-actin activated myosin ATPase activity
- Identification/characterization of proteins or small molecules that affect myosin ATPase activity
- Identification/characterization of proteins or small molecules that affect myosin / F- actin interaction
Materials
Smooth muscle myosin protein has been purified from chicken gizzards (1, 2). The full length myosin protein was purified with its essential light chains (ELC) and regulatory light chains (RLC). Myosin was then digested with papain to liberate the soluble subfragment-1 (S1) domain, which was isolated by centrifugation (3, 4). The purified myosin S1 fragment has been determined to be biologically active in an F-actin activated ATPase assay (see biological activity assay). Chicken gizzard S1 myosinprotein is supplied as a white lyophilized powder.
Storage and Resonstitution
Briefly centrifuge to collect the product at the bottom of the tube. Reconstituting a tube of CS-MYS05 with 180 ul of Milli-Q water will generate a 1.4 mg/ml stock of gizzard S1 myosin in the following buffer: 8 mM PIPES pH 7.0, 8 mM MgCl2, 5% (w/v) sucrose and1% (w/v) dextran. The protein should not be exposed to repeated freeze-thaw cycles. The lyophilized protein is stable at 4°C desiccated(<10% humidity) for 1 year.
Purity
Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 4-20% gradient polyacrylamide gel. The myosin and its light chains used to produce the myosin S1 fragment was determined to be 90% pure.
