Cat. # VCG03

Product Uses Include

• Stimulating Arp2/3 complex activity
• Isolating Arp2/3 complex from cell or tissue extract
  
  

Material
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
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).

Biological Activity
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.

Examples of publications where this product was used
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.