Cat. # AKF99

Product uses

• 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 (inquire for bulk rates)
  
  

Material
Pre-formed actin filaments (PAFs) are supplied as a lyophilized powder. PAFs have been prepared from rabbit skeletal muscle actin protein that is greater than 99% pure (Cat. # AKL99). These stringently quality controlled filaments provide highly reliable and reproducible results in assays that require actin filament substrates. The average filament length in this product is 8 µm. Using these PAFs in assays depending on filaments gives very reproducible results. In an actin filament activated myosin ATPase assay (see Fig. 1), the CV between samples (Vmax) was less than 5% and the CV of Vmax between assays using different batches of AKF99 was less than 8%.

Purity
The actin protein used to make pre-formed actin filaments is >99% pure. See Cat. # AKL99.

Biological Activity
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.

Figure 1. AKF99 activated myosin ATPase reaction. Reactions were performed using the Cytophos Endpoint Assay Kit (Cat. # BK054). Reactions contained either AKF99 (30ug) or MY03 (5ug) or MY02 (0.5ug) (Cat. # MY02 or MY03) alone or the combination of AKF99 and myosins as noted. Triplicate reactions were incubated for 20min at 37C then quenched with Cytophos reagent. Optical density readings were measured at 650nm.

Figure 2. Filament binding spin-down assay using AKF99. Greater than 90% of AKF99 (Actin) is in pellet (P) after spin-down. Alpha actinin (Cat. # AT01) binds to PAFs and ends up in pellet while BSA does not and stays in supernatant (S).