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%.
The actin protein used to make pre-formed actin filaments is >99% pure. See Cat. # AKL99.
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 (Ca t. # 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).
Ravichandran, Akshaya et al. “A Novel Actin Binding Drug with In Vivo Efficacy.” Antimicrobial agents and chemotherapy vol. 63,1 e01585-18. 21 Dec. 2018, doi:10.1128/AAC.01585-18
Kampourakis, Thomas et al. “Hypertrophic cardiomyopathy mutation R58Q in the myosin regulatory light chain perturbs thick filament-based regulation in cardiac muscle.” Journal of molecular and cellular cardiology vol. 117 (2018): 72-81. doi:10.1016/j.yjmcc.2018.02.009
Sagot, I. et al. 2002. An actin nucleation mechanism mediated by Bni1 and Profilin. Nat. Cell Biol. 4, 626 - 631.
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’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’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’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’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.
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