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The VCA (Verprolin, cofilin, acidic) domain of human WASP protein has been expressed in a bacterial expression system as a GST-tagged fusion protein. The protein has been purified by glutathione affinity chromatography and is supplied as a lyophilized powder. When reconstituted with distilled water, the complex is in the following buffer: 20 mM Tris pH 7.5, 25 mM KCl, 1 mM MgCl2, 0.5 mM EDTA, 0.2% dextran, 2% sucrose. The molecular weight of the GST-VCA domain protein is approximately 43 kDa.
Purity is determined by scanning densitometry of proteins on SDS-PAGE gels. Samples are >95% pure (Figure 1).
Figure 1: Purity determination of GST-WASP VCA domain. 20 µg of VCG03 was run on an SDS-PAGE gel and visualized by coomassie blue staining. Protein quantitation was performed using the Precision Red Protein Assay Reagent (Cat.# ADV02).
VCG03 was tested in an actin polymerization assay (Cat. # BK003). In conjunction with Arp2/3 (Cat. # RP01) it was shown to stimulate actin polymerization 20-fold compared to the control without VCG03. This indicates VCA domain specificity for stimulating the actin nucleating activity of Arp2/3.
Figure 2: Actin polymerization stimulated by Arp2/3 complex and the VCA domain of WASP. Actin polymerization was measured using kit BK003. The addition of Arp2/3 complex or the VCA domain alone to actin has minimal effects on actin polymerization, while the combination of Arp2/3 and the VCA domain strongly stimulates the rate of actin polymerization.
van der Gucht, J., Paluch, E., Plastino, J. and Sykes, C. (2005). Stress release drives symmetry breaking for actin-based movement. Proc. Natl. Acad. Sci. U. S. A. 102, 7847-7852.
Question 1: By itself, what is the effect of VCA-domain WASP protein on actin polymerization?
Answer 1: By itself, the VCA-domain WASP protein (Cat. # VCG03) partial inhibits actin polymerization. When combined with Arp2/3 (Cat. # RP01), actin polymerization is greatly enhanced. See the associated datasheets for details.
Question 2: What is the optimal concentration of Arp2/3 and VCA-domain WASP protein to modify actin polymerization?
Answer 2: We recommend that pyrene actin is present at a final concentration of 0.8 μM, Arp2/3 complex at 10 nM (Cat. # RP01) and the VCA domain of WASP at 400 nM (Cat. # VCG03). The critical factor here is the actin concentration which must be 0.80 +/-0.2 µM in the final reaction. This concentration is just below the critical concentration for assembly and hence will not polymerize on it’s own.
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