Binding Proteins

All Actin Binding Proteins (ABPs) are assayed to ensure that their activities are comparable to published values.  Assays vary by product and include F-actin severing assays, F-actin-activated ATP hydrolysis, and actin polymerization assays.  Measuring the role of WASP VCA domain protein in actin polymerization is shown (right).

Cytoskeleton, Inc. sells a range of purified actin binding proteins. All Cytoskeleton's actin binding proteins are of high purity and are rigorously tested for biological activity. Cytoskeleton also provides actin proteins, antibodies and kits for actin binding protein research.

arp-actin-pol

Actin polymerization stimulated by Arp2/3 and the WASP VCA domain.  Kit BK003 was used to study the effects of Arp2/3 (Cat. # RP01) and the WASP VCA (Cat. # VCG03) domain on actin polymerization in vitro.

Actin interacts with a multitude of proteins in the cell
It is widely known that F- and G-actin interact with a multitude of proteins in the cell (14). At present there are over 150 known actin binding proteins (ABPs), which accounts for approximately 25% of cellular protein. ABPs impart a great deal of functional diversity upon the actin cytoskeleton which is involved in processes such as muscle contraction, lamellopodia extrusion, cell locomotion, cytokinesis and cytoplasmic streaming. Investigating how these processes regulate actin filament function and dynamics is an important and ever growing field of research.

Actin binding proteins maybe isolated and studied by using biochemical, genetic and immunological approaches. Because there are over 50 types of actin binding proteins the methods used to identify them and their function/activities are also numerous and varied. Actin affinity columns are often used to affinity purify ABPs whether they are F- or G-actin binding. A simple assay to show that a potential ABP affects actin dynamics can be carried out by exploiting polymerization enhancement of pyrene actin fluorescence (see measuring actin polymer section). Studies of this nature have been instrumental in identifying novel actin binding proteins. There are several reviews worth reading on the possible approaches to identify and quantify novel actin binding proteins (15,16,17)

Cytoskeleton's actin products have been cited hundreds of times over the past 18 years. A select few are described here, for more citations on individual products please use the "Citations" tab on each individual product page. 

Alpha-actinin protein: rabbit skeletal muscle (Cat. # AT01)

van der Gucht, J., Paluch, E., Plastino, J. and Sykes, C. (2005). Stress release drives symmetry breaking for actin-based movement. Proc. Natl. Acad. Sci. U. S. A. 102, 7847-7852.
Lu, S., Carroll, S. L., Herrera, A. H., Ozanne, B. and Horowits, R. (2003). New N-RAP-binding partners α-actinin, filamin and Krp1 detected by yeast two-hybrid screening: implications for myofibril assembly. J. Cell Sci. 116, 2169-2178.
Maul, R. S., Song, Y., Amann, K. J., Gerbin, S. C., Pollard, T. D. and Chang, D. D. (2003). EPLIN regulates actin dynamics by cross-linking and stabilizing filaments. J. Cell Biol. 160, 399-407.
Karakesisoglou, I., Yang, Y. and Fuchs, E. (2000). An epidermal plakin that integrates actin and microtubule networks at cellular junctions. J. Cell Biol. 149, 195-208.
Waterman-Storer, C., Duey, D. Y., Weber, K. L., Keech, J., Cheney, R. E., Salmon, E. D. and Bement, W. M. (2000). Microtubules remodel actomyosin networks in Xenopus egg extracts via two mechanisms of F-actin transport. J. Cell Biol. 150, 361-376.

Cofilin 1 protein: human recombinant (Cat. # CF01)

van der Gucht, J., Paluch, E., Plastino, J. and Sykes, C. (2005). Stress release drives symmetry breaking for actin-based movement. Proc. Natl. Acad. Sci. U. S. A. 102, 7847-7852.
Loomis, P. A., Zheng, L., Sekerkova, G., Changyaleket, B., Mugnaini, E. and Bartles, J. R. (2003). Espin cross-links cause the elongation of microvillus-type parallel actin bundles in vivo. J. Cell Biol. 163, 1045-1055.
Vignjevic, D., Yarar, D., Welch, M. D., Peloquin, J., Svitkina, T. and Borisy, G. G. (2003). Formation of filopodia-like bundles in vitro from a dendritic network. J. Cell Biol. 160, 951-962.
Yokoo, T., Toyoshima, H., Miura, M., Wang, Y., Iida, K. T., Suzuki, H., Sone, H., Shimano, H., Gotoda, T., Nishimori, S. et al. (2003). p57Kip2 regulates actin dynamics by binding and translocating LIM-kinase 1 to the nucleus. J. Biol. Chem. 278, 52919-52923.
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.

Heavy meromyosin protein (Cat. # MH01)

Li, Y., Lin, J. L., Reiter, R. S., Daniels, K., Soll, D. R. and Lin, J. J. (2004). Caldesmon mutant defective in Ca2+-calmodulin binding interferes with assembly of stress fibers and affects cell morphology, growth and motility. J. Cell Sci. 117, 3593-3604.
Kontrogianni-Konstantopoulos, A., Huang, S. C. and Benz, E. J., Jr. (2000). A nonerythroid isoform of protein 4.1R interacts with components of the contractile apparatus in skeletal myofibers. Mol. Biol. Cell 11, 3805-3817.

Myosin II protein: rabbit skeletal muscle (Cat. # MY02)

Harris, E. S., Li, F. and Higgs, H. N. (2004). The mouse formin, FRLα, slows actin filament barbed end elongation, competes with capping protein, accelerates polymerization from monomers, and severs filaments. J. Biol. Chem. 279, 20076-20087.
Gallo, G., Yee, H. F., Jr. and Letourneau, P. C. (2002). Actin turnover is required to prevent axon retraction driven by endogenous actomyosin contractility. J. Cell Biol. 158, 1219-1228.

Profilin 1 protein: His tagged: human (Cat. # PR01)

Moseley, J. B., Sagot, I., Manning, A. L., Xu, Y., Eck, M. J., Pellman, D. and Goode, B. L. (2004). A conserved mechanism for Bni1- and mDia1-induced actin assembly and dual regulation of Bni1 by Bud6 and profilin. Mol. Biol. Cell 15, 896-907.
Vignjevic, D., Yarar, D., Welch, M. D., Peloquin, J., Svitkina, T. and Borisy, G. G. (2003). Formation of filopodia-like bundles in vitro from a dendritic network. J. Cell Biol. 160, 951-962.

Arp2/3 protein complex: bovine brain (Cat. # RP01)

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.
van der Gucht, J., Paluch, E., Plastino, J. and Sykes, C. (2005). Stress release drives symmetry breaking for actin-based movement. Proc. Natl. Acad. Sci. U. S. A. 102, 7847-7852.

WASP protein VCA domain: GST tagged: human (Cat. # VCG03)

van der Gucht, J., Paluch, E., Plastino, J. and Sykes, C. (2005). Stress release drives symmetry breaking for actin-based movement. Proc. Natl. Acad. Sci. U. S. A. 102, 7847-7852.

Question 1: Which Actin Binding Protein (ABP) is useful as an F-actin binding control? 

Answer 1: The actin binding protein alpha-actinin (Cat. # AT01) is an F-actin binding and cross-linking protein, making it useful as a control for F-actin binding studies. It's Kd is 1 µM which means that 50% binding occurs at this concentration. Therefore we aim to use alpha-actin at 2 µM as a positive control in binding assays. A 2 µM concentration of alpha-actinin is equivalent to 0.23 µg/µl in the binding reaction. Also alpha-actinin is sensitive to pH where its highest affinity occurs at pH 7.0 .

 

Question 2: Does Cytoskeleton's myosin or heavy meromyosin have F-actin motility activity? 

Answer 2:Unfortunately we do not sell motility grade myosin or heavy meromyosin for actin motility assays.  We found that quality control tests were very difficult to reproduce and therefore we could not adequately ensure the quality of reagents. Therefore the myosins (Cat. # MY02 and MY03) and the Heavy meromyosin protein (Cat. # MH01) are not recommended for F-actin motility assays. Customers who have tested these reagents have not had good success with motility and therefore we do not recommend purchasing samples to test either. If motility grade actin or heavy meromyosin is desired we recommend following the original protocol (Kron & Spudich, 1985) and freezing 100 µl aliquots in liquid nitrogen to ensure stability.

Reference: S.J. Kron  and J.A. Spudich, 1985. Fluorescent actin filaments move on myosin fixed to a glass surface. PNAS, 83, 6272-6276.

 

Please contact tservice@cytoskeleton.com with questions regarding these products. 

  1. Actin Binding Protein Spin-Down Assay Biochem Kit: human platelet actin BK013
    Actin Binding Protein Spin-Down Assay Biochem Kit: human platelet actin
    Learn More
  2. Actin Binding Protein Spin-Down Assay Biochem Kit: rabbit skeletal muscle actin BK001
    Actin Binding Protein Spin-Down Assay Biochem Kit: rabbit skeletal muscle actin
    Learn More
  3. Actin Polymerization Biochem Kit (fluorescence format): rabbit skeletal muscle actin BK003
    Actin Polymerization Biochem Kit (fluorescence format): rabbit skeletal muscle actin
    Learn More
  4. Alpha-actinin protein: rabbit skeletal muscle AT01
    Alpha-actinin protein: rabbit skeletal muscle
    Learn More
  5. G-Actin/F-actin In Vivo Assay Biochem Kit BK037
    G-Actin/F-actin In Vivo Assay Biochem Kit
    Learn More
  6. Heavy Meromyosin Protein (HMM Fragment from cardiac muscle tissue) CS-MH03

    Heavy Meromyosin Protein (HMM Fragment from cardiac muscle tissue)

    Learn More
  7. MICAL-1 Protein 6xHis MIC01
    6xHis MICAL-1 Protein Redox-CH domains (2 x 50 micrograms) Learn More
  8. MsrB2 Protein 6xHis MB201
    6xHis MsrB2 Protein aa: 42-182 (2 x 50 micrograms) Learn More
  9. Myosin II protein: rabbit skeletal muscle MY02
    Myosin II protein: rabbit skeletal muscle
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  10. Myosin protein: bovine cardiac muscle MY03
    Myosin protein: bovine cardiac muscle
    Learn More
  11. Profilin 1 protein: Untagged, human recombinant PR02
    Profilin 1 protein: Untagged, human recombinant
    Learn More
  12. Skeletal Muscle Thin Filament Complex (Rabbit Skeletal Muscle from psoas tissue) CS-TFC02

    Skeletal Muscle Thin Filament Complex (Rabbit Skeletal Muscle from psoas tissue)

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  13. Tropomyosin/Troponin Complex, bovine source, >60% pure CS-TT05
    Tropomyosin/Troponin Complex, bovine source, >60% pure
    Learn More
  14. Ultra-Precise HRV C3 Protease (human rhinovirus type 14 3C Protease) (recombinant-GST tagged) CS-UPP01

    Ultra-Precise HRV C3 Protease (human rhinovirus type 14 3C Protease)
    (recombinant-GST tagged)

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  15. WASP protein VCA domain: GST tagged: human VCG03
    WASP protein VCA domain: GST tagged: human
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