Actin protein, non-muscle (APHL99 & APHL95)

Material
Non-muscle actin has been purified from human platelets. Each unit of platelets used in the preparation of non-muscle actin has been found to be non-reactive by an FDA approved test for HBsAg, HBcAb, HIV-1/2 ab, HIV-1 RNA, HTLV I/II ab, HCV ab, HCV RNA, and syphilis. Each unit of platelets has been ALT tested with results less than an established cutoff. The isotype composition of non-muscle actin is 85% β-actin and 15% γ-actin. Non-muscle actin has an approximate molecular weight of 43 kDa. The product is provided as a lyophilized white powder. The lyophilized protein is stable for 6 months when stored desiccated to <10% humidity at 4°C. The protein should be reconstituted to 10 mg/ml with distilled water, it will then be in the following buffer: 5 mM Tris-HCl pH 8.0, 0.2 mM CaCl₂, 0.2 mM ATP, 5% sucrose, and 1% dextran.

Purity
Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 12% polyacrylamide gel. APHL99 consists of >99% pure non-muscle actin while APHL95 is >95% pure (see Figure 1).

Figure 1: Figure 1. Purities of human platelet non-muscle actin protein. 100 µg of >99% pure (APHL99) and >95% pure (APHL95) non-muscle actin were run on SDS-PAGE gels and stained with coomassie blue. The arrow indicates actin protein (~43 kDa), the arrowhead a gelsolin contaminant (~90 kDa). The minor impurities in the purified actins are predominantly actin binding proteins such as gelsolin and α-actinin. Protein quantitation was determined with the Precision Red Protein Assay Reagent (Cat. # ADV02)

Biological Activity
The biological activity of muscle actinis determined by its ability to efficiently polymerize into filaments (F-actin) in vitro and separate from unpolymerized components in a spin down assay. Stringent quality control ensures that APHL99 produces >85% F-actin and APHL95 produces >75% F-actin in this assay.

Examples of publications where these products were used
Chew, C. S., Chen, X., Parente, J. A., Jr., Tarrer, S., Okamoto, C. and Qin, H. Y. (2002). Lasp-1 binds to non-muscle F-actin in vitro and is localized within multiple sites of dynamic actin assembly in vivo. J. Cell Sci. 115, 4787-4799

Gohla, A., Birkenfeld, J. and Bokoch, G. M. (2005). Chronophin, a novel HAD-type serine protein phosphatase, regulates cofilin-dependent actin dynamics. Nat. Cell Biol. 7, 21-29.

Kessels, M. M., Engqvist-Goldstein, A. E. and Drubin, D. G. (2000). Association of mouse actin-binding protein 1 (mAbp1/SH3P7), an Src kinase target, with dynamic regions of the cortical actin cytoskeleton in response to Rac1 activation. Mol. Biol. Cell 11, 393-412.

Kuriyama, R., Gustus, C., Terada, Y., Uetake, Y. and Matuliene, J. (2002). CHO1, a mammalian kinesin-like protein, interacts with F-actin and is involved in the terminal phase of cytokinesis. J. Cell Biol. 156, 783-790.

Posern, G., Miralles, F., Guettler, S. and Treisman, R. (2004). Mutant actins that stabilise F-actin use distinct mechanisms to activate the SRF coactivator MAL. EMBO J. 23, 3973-3983.

Roger, B., Al-Bassam, J., Dehmelt, L., Milligan, R. A. and Halpain, S. (2004). MAP2c, but not tau, binds and bundles F-actin via its microtubule binding domain. Curr. Biol. 14, 363-371.

Vartiainen, M., Ojala, P. J., Auvinen, P., Peranen, J. and Lappalainen, P. (2000). Mouse A6/twinfilin is an actin monomer-binding protein that localizes to the regions of rapid actin dynamics. Mol. Cell. Biol. 20, 1772-1783.

Zhou, D., Mooseker, M. S. and Galan, J. E. (1999). Role of the S. typhimurium actin-binding protein SipA in bacterial internalization. Science 283, 2092-2095.