Pre-formed actin filaments (rabbit skeletal muscle)

On Arrival: 4°C

Question 1: What is the isotype composition of the skeletal muscle actin?

Answer 1: Skeletal muscle actin (CAt. #AKF99) is composed of 99.5% alpha-actin skeletal muscle isoform (Swiss Protein Databank Acc. # P68135.1) .

Question 2: Can the pre-formed actin filaments from rabbit skeletal muscle be used with cardiac myosin from bovine to study myosin&rsquo, s ATPase activity?

Answer 2: Yes, the pre-formed actin filaments prepared from rabbit skeletal muscle (CAt. #AKF99) is recommended to activate the ATPase activity of myosin prepared from bovine cardiac muscle ((Cat. #MY03). The activation of cardiac muscle myosin is much less than the activation observed with rabbit skeletal myosin (Cat. #MY02), i.e. MY03 atpase is activated 3 to 4 fold over it&rsquo, s endogenous, whereas MY02 is activated 50 to 100 fold.

Question 3: Is this product suitable for high resolution microscopic imaging of actin filaments?

Answer 3: No, the pre-formed actin filaments are ideal for activating myosin&rsquo, s ATPase activity, and spin-down binding assays but we do not recommend AKF99 for high resolution microscopic analysis. The reason is that AKF99 preparation involves cycles of lyophilization and freeze/thaw which create denatured zones in the protein. The ability to activate myosin&rsquo, s ATPase activity or bind F-actin accessory proteins is not affected. For high resolution microscopy, we recommend using 99% pure actin monomers (Cat. #AKL99) to polymerize fresh filaments and use them for such analyses.

Question 4: Upon resuspension, what are the buffer components?

Answer 4: The protein will then be in the following buffer: 5 mM Tris-HCl pH 8.0, 0.2 mM CaCl₂, 0.2 mM ATP, 5 mM MgCl₂ and 5% (w/v) sucrose.

Substrate for activation of myosin ATPases (see Fig. 1)
Substrate for discovery and characterization of actin binding proteins (ABPs) (see Fig. 2)
Determination of IC50s for myosin inhibitors
Used in Cytoskeleton, Inc's ATPase assays (Cat. #BK051 and BK054)
Ideal for HTS applications

PAFs serve as a substrate for myosin motor proteins. Myosin motor proteins orchestrate a wide range of kinetic events within a cell. They have been shown to move cargoes such as vesicles along actin filament tracks. Myosins operate by utilizing the energy of ATP to hydrolysis, an activity that is greatly enhanced in the presence of PAFs. A PAF activated Enzyme Linked Inorganic Phosphate ATPase (ELIPA) assay (Cat. #BK051 and BK054) can therefore be used as a test for the biological activity of AKF99 (Fig. 1). The motor protein used in this assay is myosin motor protein (Myosin II: Cat. # MY02 or MY03). AKF99 is also an excellent substrate for detecting actin binding proteins, see Fig. 2.