Arp2/3 Protein Complex (Bovine brain)

Question 1: What is the best way to store Arp2/3 (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 &mu, l 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 (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 (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 &mu, M, the Arp2/3 complex is at 10 nM and the VCA domain ((Cat. #VCG03) is at 400 nM.