Product Uses Include
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.
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)
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.
K. Sobierajska et al., 2014. PDI directly interacts with β-actin Cys374 and regulates cytoskeleton reorganization. J. Biol. Chem. doi: 10.1074/jbc.M113.479477.
Lamothe et al., 2013. AK1 is essential for osteoclast differentiation and is an important modulator of cell death by apoptosis and necroptosis. Mol. Cell. Biol. 33, 582-595.
Gohla et al., 2005. Chronophin, a novel HAD-type serine protein phosphatase, regulates cofilin-dependent actin dynamics. Nat. Cell Biol. 7, 21-29.
Posern et al., 2004. Mutant actins that stabilise F-actin use distinct mechanisms to activate the SRF coactivator MAL. EMBO J. 23, 3973-3983.
Roger et al., 2004. MAP2c, but not tau, binds and bundles F-actin via its microtubule binding domain. Curr. Biol. 14, 363-371.
Searles et al., 2004. Actin cytoskeleton organization and poststranscriptional regulation of endothelial nitric oxide synthase during cell growth. Circulation Research 95 ,488-495.
Chew et al., 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
Kuriyama et al., 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.
Kessels et al., 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.
Vartiainen et al., 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 et al., 1999. Role of the S. typhimurium actin-binding protein SipA in bacterial internalization. Science 283, 2092-2095.
Question 1: Do you have pyrene-labeled non-muscle actin for use in a polymerization assay?
Answer 1: Pyrene-labeled non-muscle actin has been shown to be unstable under normal storage conditions and was discontinued. To examine the polymerization of unlabeled non-muscle actin, please click here for a polymerization protocol that uses an excess of unlabeled non-muscle actin (Cat# APHL99) + a small amount of pyrene-labeled muscle actin (Cat. # AP05). The pyrene muscle actin will not polymerize efficiently on its own at the concentration used in this assay, so the reaction is dependent on unlabeled actin polymerization for F-actin formation. In this way, the pyrene-labeled muscle actin is taken up and polymerized to serve as a reporter for polymerization of the unlabeled non-muscle actin that is present at a much greater concentration.
Question 2: Are the actin products shipped as pure G-actin or a mixture of G- and F-actin?
Answer 2: Most of our actin proteins are sold in the monomer form (G-actin) because this is stable to freezing and lyophilization. That being said, on the day of the experiment, we do recommend incubating the actin on ice for 60 min before beginning the experiment to depolymerize any actin oligomers that might have formed during storage. Typically actin is first diluted to 0.4 or 0.2 mg/ml concentration and then this can be incubated on ice for 60 min to depolymerize any actin oligomers that might have formed. If you are working with an actin concentration above 0.4 mg/ml, we recommend the ice incubation followed by a high-speed centrifugation (100,000 x g) for 60 min to pellet any actin oligomers that may not have depoymerized. Remove the top 80% of the supernatant and use this as your G-actin stock. We also provide pre-formed actin filaments (Cat. # AKF99) that are shipped lyophilized and upon resuspension, the filaments are ready for use and average 5-10 microns in length.
If you have any questions concerning this product, please contact our Technical Service department at firstname.lastname@example.org