Cat. # AP05
Product Uses Include
- Studying actin polymerization in vitro.
- Studying the effects of actin binding proteins and drugs on actin polymerization in vitro.
Material
Purified rabbit muscle actin (Cat. # AKL99) has been modified to contain covalently linked pyrene at the cysteine 374 residue. An N-(1-pyrene) iodoacetamide is used to label the actin protein. Pyrene labeling stoichiometry has been determined to be 0.6 dyes per actin monomer. Pyrene labeled rabbit muscle actin has an approximate molecular weight of 43 kDa, and is supplied as a white 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 20 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 4-20% gradient polyacrylamide gel. Pyrene muscle actin is >99% pure (see Figure 1).
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Figure 1. Pyrene Muscle Actin Protein Purity Determination. A 100 µg sample of pyrene muscle actin (molecular weight approx. 43 kDa) was separated by electrophoresis in a 4-20% SDS-PAGE system, and stained with Coomassie Blue. Protein quantitation was determined with the Precision Red Protein Assay Reagent (Cat. # ADV02). |
Biological Activity
The fluorescent signal of monomer pyrene actin is enhanced during its polymerization into filaments, making it an ideal tool for monitoring actin filament formation. Stringent quality control ensures that AP05 pyrene F-actin has a 7-12 fold fluorescent enhancement over non-polymerized pyrene G-actin (See Fig 2).
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Figure 2. Fluorescence enhancement during pyrene actin polymerization. Pyrene muscle actin was polymerized in duplicate wells of a 96-well plate by the addition of Actin Polymerization Buffer (Cat. # BSA02). The fluorescent signal was scanned every 30 s for 1 h. Polymerized pyrene F-actin shows a 10 fold fluorescent enhancement over non-polymerized pyrene G-actin and buffer control. |
Examples of publications where this product was used
Balcer, H. I., Goodman, A. L., Rodal, A. A., Smith, E., Kugler, J., Heuser, J. E. and Goode, B. L. (2003). Coordinated regulation of actin filament turnover by a high-molecular-weight Srv2/CAP complex, cofilin, profilin, and Aip1. Curr. Biol. 13, 2159-2169.
Duncan, M. C., Cope, M. J., Goode, B. L., Wendland, B. and Drubin, D. G. (2001). Yeast Eps15-like endocytic protein, Pan1p, activates the Arp2/3 complex. Nat. Cell Biol. 3, 687-690.
Humphries, C. L., Balcer, H. I., D'Agostino, J. L., Winsor, B., Drubin, D. G., Barnes, G., Andrews, B. J. and Goode, B. L. (2002). Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin. J. Cell Biol. 159, 993-1004.
Leng, Y., Zhang, J., Badour, K., Arpaia, E., Freeman, S., Cheung, P., Siu, M. and Siminovitch, K. (2005). Abelson-interactor-1 promotes WAVE2 membrane translocation and Abelson-mediated tyrosine phosphorylation required for WAVE2 activation. Proc. Natl. Acad. Sci. U. S. A. 102, 1098-1103.
Uruno, T., Liu, J., Zhang, P., Fan, Y., Egile, C., Li, R., Mueller, S. C. and Zhan, X. (2001). Activation of Arp2/3 complex-mediated actin polymerization by cortactin. Nat. Cell Biol. 3, 259-266.
Wahlstrom, G., Vartiainen, M., Yamamoto, L., Mattila, P. K., Lappalainen, P. and Heino, T. I. (2001). Twinfilin is required for actin-dependent developmental processes in Drosophila. J. Cell Biol. 155, 787-796.
| Product description | Cat. # | Amount | Price & Order |
| Pyrene actin protein | AP05-A | 1 x1 mg |
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| AP05-B | 5 x 1 mg |
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| AP05-XL | Large quantities |
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