Cat. # RP01

Product Uses Include

• Positive control for in vitro actin polymerization stimualting compounds
• Discovery of Arp2/3 activating or inhibiting compound
• Characterization and discovery of Arp2/3 interacting proteins
  
  

Material
The Arp2/3 protein complex consists of 7 protein subunits, all present in approximately equal stoichiometry (see Figure 1). The Arp2/3 complex is a key regulator of actin filament nucleation and the protein subunits are highly evolutionarily conserved.  The protein complex has been purified from bovine brain 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, 0.1 mM ATP, 0.2% dextran and 2% sucrose.  The molecular weight of the Arp2/3 complex is 224 kDa.

Purity
Purity is determined by scanning densitometry of proteins on SDS-PAGE gels.  Samples are >95% pure (Figure 1).

Figure 1. Arp2/3 complex purity determination. 10 µg of RP01 was run on an SDS-PAGE gel and proteins were visualized by coomassie staining.  Protein quantitation was performed using the Precision Red Protein Assay Reagent (Cat.# ADV02).

Biological Activity
RP01 was tested in an actin polymerization assay (Cat. # BK003). In conjunction with the VCA domain of WASP (Cat. # VCG03), an Arp2/3 activator, it was shown to stimulate actin polymerization by 20-fold compared to the control without RP01. This indicates Arp 2/3 specificity for actin polymerization induction (Figure 2).

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
Leng, Y., Zhang, J., Badour, K., Arpaia, E., Freeman, S., Cheung, P., Siu, M. and Siminovitch, K. (2005). Abelson-interactor-1 promotes WAVE2 membrane translocation and Abelson-mediated tyrosine phosphorylation required for WAVE2 activation. Proc. Natl. Acad. Sci. U. S. A. 102, 1098-1103.

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.