Product Uses
- Measurement of calcium activated cardiac HMM ATPase activity when bound to thin filaments.
- Identification/characterization of proteins or small molecules that affect the TT complex regulation and cardiac HMM ATPase activity
- Identification/characterization of proteins or small molecules that affect cardiac HMM / F- actin interaction
Materials
Cardiac myosin protein has been purified from bovine heart tissue (1,2,3). The full length myosin protein was purified with its essential light chains (ELC) and regulatory light chains (RLC), see Figure 1 and 2. Myosin was then digested with a-chymotrypsin in the presence of MgCl2 to liberate the soluble heavy fragment (HMM) domain, which was isolated by centrifugation followed by anionic exchange chromatography to remove S1 (2,3). The purified myosin HMM fragment has been determined to be biologically active in an F-actin activated ATPase assay (see biological activity assay). Bovine cardiac myosin S1 fragment protein is supplied as a white lyophilized powder.
Storage and Resonstitution
Briefly centrifuge to collect the product at the bottom of the tube. Reconstituting a 100 µg tube of MH03 with 100 ml of 10 mM Tris-HCl pH 7.50, 30 mM KCl, 1 mM EDTA, 1 mM DTT in Milli-Q water The protein should not be exposed to repeated freeze-thaw cycles. The lyophilized protein is stable at 4°C desiccated (<10% humidity) for 6 months.
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 (see Figure 2). After chymotrypsin digestion and FPLC the HMM and light chains constitute approx. 85% of total protein. The S1 myosin content is less than 1%.
