Rhodamine actin, non-muscle (APHR)

Product Uses Include

In vivo actin polymerization studies (micro-injection into non-muscle cells)
In vitro motility studies using fluorescent F-actin and non-muscle myosins

Material
Non-muscle actin purified from human platelets (Cat. # APHL99) has been modified to contain covalently linked rhodamine at random surface lysine residues. An activated ester of rhodamine is used to label the protein. The labeling stoichiometry has been determined to be 0.4 dyes per actin monomer. Rhodamine non-muscle actin has an approximate molecular weight of 43 kDa. Rhodamine non-muscle actin is supplied as a pink lyophilized 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.

Rhodamine actin from a muscle source is also available (Cat. # AR05).

Purity
Protein purity is determined by scanning densitometry of Coomassie Blue stained protein on a 12% polyacrylamide gel. APHR rhodamine non-muscle actin is >99% pure (see Figure 1). No free dye is detectable in the final product

Figure 1. Rhodamine Non-Muscle Actin Protein Purity Determination. A 100 µg sample of rhodamine non-muscle actin (molecular weight approx. 43 kDa) was separated by electrophoresis in a 12% SDS-PAGE system, and stained with Coomassie Blue. Minor protein bands present at 86 and 120 kDa are dimers and trimers of rhodamine actin respectively, and constitute <1% of the total protein. Protein quantitation was performed with the Precision Red Protein Assay Reagent (Cat. # ADV02).

Biological activity
The biological activity of rhodamine non-muscle actin is determined by its ability to efficiently polymerize into filaments in vitro and separate from unpolymerized components in a spin down assay. Stringent quality control ensures that >80% of the labeled non-muscle actin polymerizes in this assay, which is comparable to the unconjugated protein (APHL99)

Examples of publications where this product was used
Idrissi, F. Z., Wolf, B. L. and Geli, M. I. (2002). Cofilin, but not profilin, is required for myosin-I-induced actin polymerization and the endocytic uptake in yeast. Mol. Biol. Cell 13, 4074-4087.

Machesky, L. M. and Hall, A. (1997). Role of actin polymerization and adhesion to extracellular matrix in Rac- and Rho-induced cytoskeletal reorganization. J. Cell Biol. 138, 913-926.

Smilenov, L. B., Mikhailov, A., Pelham, R. J., Marcantonio, E. E. and Gundersen, G. G. (1999). Focal adhesion motility revealed in stationary fibroblasts. Science 286, 1172-1174.

Tsukada, M., Prokscha, A., Ungewickell, E. and Eichele, G. (2005). Doublecortin association with actin filaments is regulated by neurabin II. J. Biol. Chem. 280, 11361-11368.

Vasanji, A., Ghosh, P. K., Graham, L. M., Eppell, S. J. and Fox, P. L. (2004). Polarization of plasma membrane microviscosity during endothelial cell migration. Dev. Cell 6, 29-41.