NOTE: This product has been replaced by the porcine version of Arp2/3 protein complex. See Cat. # RP01P.
Product Uses Include
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, 1% dextran and 5% sucrose. The molecular weight of the Arp2/3 complex is 224 kDa.
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).
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.
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.
Question 1: What is the best way to store Arp2/3 (Cat. # RP01)?
Answer 1: Store the lyophilized Arp2/3 protein desiccated (<10% humidity) at 4°C where it is stable for 6 months. Lyophilized protein can also be stored desiccated at -70°C where it will be stable for 6 months. However, at -70°C the rubber seal in the lid of the tube could crack and allow in moisture. Therefore we recommend storing at 4°C. If stored at -70°C, it is imperative to include desiccant with the lyophilized protein if this storage condition is utilized. Resuspend the protein complex to 5 mg/ml with 10 μl of cold Milli-Q water. When resuspended, 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, 1.0% (v/v) dextran and 5% (v/v) sucrose. The protein should then be aliquoted into experiment-sized amounts, snap frozen in liquid nitrogen and stored at -70°C where it is stable for 6 months. Further dilution of Arp2/3 should be made in the following buffer: 20 mM Tris pH 7.5, 25 mM KCl, 1 mM MgCl2 and 1 mM DTT (Note: add DTT to the buffer immediately prior to use). NOTE: It is very important to snap freeze the Arp2/3 protein in liquid nitrogen as other methods of freezing will result in significantly reduced activity. Defrost rapidly by placing in a room temperature water bath for 1 min. Avoid repeated freeze/thaw cycles.
Question 2: Is the Arp2/3 (Cat. # RP01) compatible with the actin polymerization kit (Cat. # BK003)?
Answer 2: Yes, Arp2/3 works very well with the actin polyermization kit (Cat. # BK003) to study the effects of this actin binding protein on actin polymerization. Please see the Arp2/3 datasheet (Cat. # RP01) for additional information. The Arp2/3 complex is able to induce the branched polymerization of actin filaments in vitro at a molar ratio of 1:200 (Arp2/3:actin). This stimulation is observable in an in vitro polymerization assay; however, the stimulation from Arp2/3 alone is very low under typical polymerization conditions. In the presence of N-WASP protein (or the VCA domain of N-WASP, Cat. # VCG03), the nucleating activity of Arp2/3 is greatly enhanced. In the polymerization assay described in the Arp2/3 datasheet, pyrene actin (Cat. # AP05 or BK003) is present at a final concentration of 0.8 μM, the Arp2/3 complex is at 10 nM and the VCA domain (Cat. # VCG03) is at 400 nM.
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