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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 porcine 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.

Note: RP01P from procine brain replaces RP01 from bovine brain. We have compared the activity of Arp2/3 complex from both sources and have found them to behave identically in actin polymerization assays (see Fig. 2)

Purity：

       Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 4-20% polyacrylamide gel. Arp2/3 protein complex is determined to be 90% pure (see Figure 1).

       Figure 1. Arp2/3 Protein Purity Determiniation: A 10 μg sample of Arp2/3 complex was separated by electro-phoresis in a 4-20% SDS-PAGE system and stained with Coomassie Blue. Protein quantita-tion was determined with the Precision RedTM Protein Assay Reagent (Cat. # ADV02). Mark12 molecular weight markers are from Life Technologies.

Biological Activity：
       RP01P 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 Assay with Arp2/3 and VCA domain proteins: Actin polymerization was carried out as described in the method; All reactions contain 0.8 μM actin. Reactions containing Arp2/3 or VCA (data not shown) show little or no enhancement of actin nucleation. Actin alone also shows low polymerization com-petence under these conditions (Actin alone reactions). In the presence of the VCA domain however, Arp2/3 results in an en-hancement of actin nucleation (Note the steep nucleation phase of polymerization). Actin polymerization is measured in arbitrary fluorescent units over time. In our tests porcine derived Arp2/3(wells E & F) behaved identically to Arp2/3 derived from bovine sources (wells G & H).