Product Uses
- Measurement of microtubule-activated ATPase/GTPase assays.
- Identification/characterization of proteins or small molecules that affect motor ATPase/GTPase activity.
- Identification/characterization of proteins or small molecules that affect motor/microtubule interactions.
Material
Cytoplasmic dynein was purified from porcine brain by the procedure of B.M. Paschal et al. (1). The preparation contains dynein heavy chain (520 kDa), intermediate chain (54, 59, and 74 kDa) and light chains (c.20 kDa). The protein has been determined to be biologically active in a microtubule-activated ATPase assay (see below). The protein is supplied as a lyophilized white powder.
Storage and Reconstitution
The protein is supplied in 50 µg amounts which are designed for single use experiments. The powder is resuspended on ice with 50 µl of ice cold water to create a 1 mg/ml solution of dynein with a final buffer composition of 20 mM Tris-HCl pH 7.5, 50 mM KCl, 2 mM MgCl2, 0.5 mM EGTA plus 20% (w/v) sucrose. In this formulation the dynein will remain active for 3 days when stored at 4°C. Do not refreeze the protein as this will irreversibly denature the protein.
Purity
Protein purity is determined by scanning densitometry of a Coomassie-stained SDS-PAGE gradient gel. Figure 1, lane 9 shows 10 mg of CS-DN01 protein and purity was determined to be c.80%. The total protein in each tube will therefore be approximately 20% greater than the amount shown on the tube. Contaminants include kinesin, dynamin, dynactin and tubulin.