Product Uses
- Measurement of calcium activated myosin ATPase activity when bound to thin filaments.
- Identification/characterization of proteins or small molecules that affect the TT complex regulation and myosin ATPaseactivity
- Identification/characterization of proteins or small molecules that affect myosin / F-actin interaction
Material
The Tropomyosin / Troponin protein (TT) complex has been purified from bovine cardiac muscle (1). The TT complex is composed of five proteins Tropomyosin alpha: Tropomyosin beta: TroponinC : Troponin I : Troponin T in a stoichiometric ratio of 1:1:1:1:1, see Figure 1. The complex has been determined to be biologically active in an F-actin / calcium activated myosin ATPase assay (see biological activity assay). The complex is supplied as a white lyophilized powder.
Storage and Reconstitution
Briefly centrifuge to collect the product at the bottom of the tube. The protein should be reconstituted to 3 mg/ml by the addition of ice cold Milli-Q water. The protein will be in the following buffer: 20 mM PIPES pH 7.0, 25 mM KCl, 5% (w/v) sucrose and 1% (w/v) dextran. In order to maintain high biological activity of the protein, it is recommended that the protein solution be aliquoted into "experiment sized" amounts, snap frozen in liquid nitrogen and stored at -70°C. The protein is stable for 6 months if stored at -70°C. 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 TT complex was determined to be 85% pure. Antibodies against each component were used to verify each one was present
