Product Uses Include
- Identification and characterization of non-muscle actin binding proteins
- In vitro actin polymerization studies
- Antibody standard for Western blot analysis
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% ?-actin and 15% ?-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 4C. 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 ug 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 (ca.43 kDa), the arrowhead a gelsolin contaminant (ca.90 kDa). The minor impurities in the purified actins are predominantly actin binding proteins such as gelsolin and ?-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.