Actin Assays
Cytoskeleton's Biochem Kits™ are comprehensive kits for assaying different aspects of cytoskeletal biochemistry and signal transduction. These kits usually form the basis of a figure in a publication, please check the citations tab for examples. The kits come with all the reagents needed for your assay as well as detailed instruction on how to use them, so you will be ready to do your assays as soon as you have the kits!
- Measure the effects of proteins and drugs on actin polymerization/depolymerization.
- Discover and characterize actin binding proteins (ABPs).
- Quantitate G-actin (monomer) vs F-actin (polymer) in cell or tissue lysate.
- Minimize set-up time from months to days.
Many publications cite the use of Cytoskeleton's kits in the Materials and Methods section of papers. Usually the citation is associated with a particular result in the form of a graph or image that helps the you, the authors, present your findings. This indicates the utility of the Kits to produce publication quality data in a short timeframe thus helping improve the productivity of your efforts. Example citations for actin kits are shown below. More citations are available on individual product pages.
Actin Binding Protein Spin-Down Assay Biochem Kit: rabbit skeletal muscle actin (Cat. # BK001) |
| Takeshita, N., Ohta, A. and Horiuchi, H. (2005). CsmA, a class V chitin synthase with a myosin motor-like domain, is localized through direct interaction with the actin cytoskeleton in Aspergillus nidulans. Mol. Biol. Cell 16, 1961-1970. |
| Banerjee, J. and Wedegaertner, P. B. (2004). Identification of a novel sequence in PDZ-RhoGEF that mediates interaction with the actin cytoskeleton. Mol. Biol. Cell 15, 1760-1775. |
| Kumar, N., Zhao, P., Tomar, A., Galea, C. A. and Khurana, S. (2004). Association of villin with phosphatidylinositol 4,5-bisphosphate regulates the actin cytoskeleton. J. Biol. Chem. 279, 3096-3110. |
| Torgler, C. N., Narasimha, M., Knox, A. L., Zervas, C. G., Vernon, M. C. and Brown, N. H. (2004). Tensin stabilizes integrin adhesive contacts in Drosophila. Dev. Cell 6, 357-369. |
| Oliver, C. J., Terry-Lorenzo, R. T., Elliott, E., Bloomer, W. A., Li, S., Brautigan, D. L., Colbran, R. J. and Shenolikar, S. (2002). Targeting protein phosphatase 1 (PP1) to the actin cytoskeleton: the neurabin I/PP1 complex regulates cell morphology. Mol. Cell. Biol. 22, 4690-4701. |
| Hildebrand, J. D. and Soriano, P. (1999). Shroom, a PDZ domain-containing actin-binding protein, is required for neural tube morphogenesis in mice. Cell 99, 485-497. |
Actin Polymerization Biochem Kit (fluorescence format): rabbit skeletal muscle actin (Cat. # BK003) |
| Takamiya, R., Takahashi, M., Park, Y. S., Tawara, Y., Fujiwara, N., Miyamoto, Y., Gu, J., Suzuki, K. and Taniguchi, N. (2005). Overexpression of mutated Cu,Zn-SOD in neuroblastoma cells results in cytoskeletal change. Am. J. Physiol. 288, C253-259. |
| Kumar, N., Tomar, A., Parrill, A. L. and Khurana, S. (2004). Functional dissection and molecular characterization of calcium-sensitive actin-capping and actin-depolymerizing sites in villin. J. Biol. Chem. 279, 45036-45046. |
| Fontao, L., Geerts, D., Kuikman, I., Koster, J., Kramer, D. and Sonnenberg, A. (2001). The interaction of plectin with actin: evidence for cross-linking of actin filaments by dimerization of the actin-binding domain of plectin. J. Cell Sci. 114, 2065-2076. |
| Zhai, L., Zhao, P., Panebra, A., Guerrerio, A. L. and Khurana, S. (2001). Tyrosine phosphorylation of villin regulates the organization of the actin cytoskeleton. J. Biol. Chem. 276, 36163-36167. |
| Blader, I. J., Cope, M. J., Jackson, T. R., Profit, A. A., Greenwood, A. F., Drubin, D. G., Prestwich, G. D. and Theibert, A. B. (1999). GCS1, an Arf guanosine triphosphatase-activating protein in Saccharomyces cerevisiae, is required for normal actin cytoskeletal organization in vivo and stimulates actin polymerization in vitro. Mol. Biol. Cell 10, 581-596. |
Actin Binding Protein Spin-Down Assay Biochem Kit: human platelet actin (Cat. # BK013) |
| 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. |
| Falcon-Perez, J. M., Starcevic, M., Gautam, R. and Dell'Angelica, E. C. (2002). BLOC-1, a novel complex containing the pallidin and muted proteins involved in the biogenesis of melanosomes and platelet-dense granules. J. Biol. Chem. 277, 28191-28199. |
| Zhai, L., Zhao, P., Panebra, A., Guerrerio, A. L. and Khurana, S. (2001). Tyrosine phosphorylation of villin regulates the organization of the actin cytoskeleton. J. Biol. Chem. 276, 36163-36167. |
| Leung, C. L., Sun, D., Zheng, M., Knowles, D. R. and Liem, R. K. (1999). Microtubule actin cross-linking factor (MACF): a hybrid of dystonin and dystrophin that can interact with the actin and microtubule cytoskeletons. J. Cell Biol. 147, 1275-1286. |
G-Actin/F-actin In Vivo Assay Biochem Kit (Cat. # BK037) |
| Rapier et al., 2010. Cancer Cell Int. 10, 24 |
| Rapier et al. (2010). The extracellular matrix microtopography drives critical changes in cellular motility and Rho A activity in colon cancer cells. Cancer Cell International 10 ,24. |
| Meeks, M. K., Ripley, M. L., Jin, Z. and Rembold, C. M. (2005). Heat shock protein 20-mediated force suppression in forskolin-relaxed swine carotid artery. Am. J. Physiol. 288, C633-639. |
| Zhang, W., Wu, Y., Du, L., Tang, D. D. and Gunst, S. J. (2005). Activation of the Arp2/3 complex by N-WASp is required for actin polymerization and contraction in smooth muscle. Am. J. Physiol. 288, C1145-1160. |
| Chen, G., Raman, P., Bhonagiri, P., Strawbridge, A. B., Pattar, G. R. and Elmendorf, J. S. (2004). Protective effect of phosphatidylinositol 4,5-bisphosphate against cortical filamentous actin loss and insulin resistance induced by sustained exposure of 3T3-L1 adipocytes to insulin. J. Biol. Chem. 279, 39705-39709. |
| Tang, D. D. and Gunst, S. J. (2004). The small GTPase Cdc42 regulates actin polymerization and tension development during contractile stimulation of smooth muscle. J. Biol. Chem. 279, 51722-51728. |
| Searles CD, Ide L, Davis ME, Cai H, Weber M (2004). Actin cytoskeleton organization and poststranscriptional regulation of endothelial nitric oxide synthase during cell growth. Circulation Research 95 ,488-495. |
| Tu, Y., Wu, S., Shi, X., Chen, K. and Wu, C. (2003). Migfilin and Mig-2 link focal adhesions to filamin and the actin cytoskeleton and function in cell shape modulation. Cell 113, 37-47. |
Question 1: Which kit is the best choice for detecting actin binding proteins?
Answer 1: Cytoskeleton has two actin binding protein spin-down assay kits that are ideal for identifying and characterizing actin binding proteins. One kit uses muscle actin (Cat. # BK001) and another kit uses non-muscle actin (Cat. # BK013). Both assays utilize co-sedimentation and western blotting to identify if a protein binds to monomeric (localized to supernatant) or filamentous actin (localized to pellet) and whether the protein has F-actin severing or bundling properties. The protein(s) of interest will need to be detected by visualization with an antibody to the protein itself or a tag on the protein. Actin distribution is also determined with an included anti-actin antibody.
Question 2: How do I measure actin polymerization with the pyrene assay when there is no available pyrene conjugate for the actin I'm interested in (i.e., cardiac, smooth muscle or cytoplasmic actins)?
Answer 2: To examine the polymerization of actin that is unlabeled, please click here for a polymerization protocol that uses an excess of unlabeled actin (cardiac, smooth muscle or cytoplasmic) + a small amount of pyrene-labeled muscle actin. 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 actin that is present at a much greater concentration.
Question 3: How do I measure G- and F-actin from cell or tissue extracts?
Answer 3: The most reproducible and accurate method of determining the amount of free globular-actin (G-actin) content versus filamentous actin (F-actin) content in cell or tissue samples is to use Western blot quantitation of F-actin and G-actin fractions. The general approach is to homogenize cells or tissue in F-actin stabilization buffer, followed by a high-speed centrifugation to separate the F-actin from the G-actin pool. The fractions are then separated by SDS-PAGE and actin is quantitated by western blot. The final result gives the most accurate method of determining the ratio of F-actin incorporated into the cytoskeleton versus the G-actin found in the cytosol. Cytoskeleton offers a straightforward and easy option to quantify G- and F-actin with the G-actin/F-actin In Vivo Assay Kit (Cat. # BK037). This kit provides all of the necessary reagents to quantify levels of monomeric and polymeric actin in cell or tissue samples under different treatment conditions. Samples are centrifuged and then pellets (F-actin) and supernatants (G-actin) are solubilized in SDS loading buffer and processed by western blotting.
For more information, please contact tservice@cytoskeleton.com with questions regarding these products.
- Actin Binding Protein Spin-Down Assay Biochem Kit: rabbit skeletal muscle actin BK001Learn MoreActin Binding Protein Spin-Down Assay Biochem Kit: rabbit skeletal muscle actin
- Actin Polymerization Biochem Kit (fluorescence format): rabbit skeletal muscle actin BK003Learn MoreActin Polymerization Biochem Kit (fluorescence format): rabbit skeletal muscle actin
- F-actin Visualization Biochem Kit (fluorescence format) BK005Learn MoreF-actin Visualization Biochem Kit (fluorescence format)
- Actin Binding Protein Spin-Down Assay Biochem Kit: human platelet actin BK013Learn MoreActin Binding Protein Spin-Down Assay Biochem Kit: human platelet actin
- G-Actin/F-actin In Vivo Assay Biochem Kit BK037Learn MoreG-Actin/F-actin In Vivo Assay Biochem Kit