Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin
Jyoti Srivastava, Diane Barber
Department of Cell and Tissue Biology, University of California, San Francisco
Citation: Srivastava J., Barber D. (2008). Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin. JoVE. 13. http://www.jove.com/index/Details.stp?ID=690, doi:
The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates
tension and helps maintains cellular shape. Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly
remodeling. A number of proteins can bind to the actin cytoskeleton. The binding of a particular protein to F-actin is often desired to support cell
biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is
an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin. The basic principles of the assay involve
an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein
co-sedimenting with F-actin. Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition
1. Preparation of actin
The source of actin used in this assay was non-muscle human platelets (ß-actin) obtained from Cytoskeleton Inc. Stock aliquots at 10 mg/ml are
kept in the -70ºC freezer. For the assay, the actin is diluted to 0.4 mg/ml in a buffer containing 5 mM Tris pH 8, 0.2 mM CaCl2, 0.2 mM ATP and
0.5 mM DTT, centrifuged at 20,000g for 10 mins at 4ºC. The supernatant, which is monomeric actin is ready to be polymerized. Actin
polymerization is induced by the addition 50 mM KCl, 1 mM ATP and 2 mM MgCl2. The polymerization occurs at room temperature for 1 hour.
2. Preparation of protein
In this assay, we are using the C-terminus of talin, which has been purified as a GST-tagged protein. The protein to be used in the assay is
subjected to a high-speed centrifugation to pre-clear of aggregates prior to incubation with F-actin.
The protein is spun at 100,000g in a Beckman ultracentrifuge for 20 mins at 22ºC.
3. Actin-protein Incubation
The amount of actin and protein in an assay will vary. In this example we are using actin in excess. Normally, a molar ratio is calculated, eg. a 4:1
molar ratio of actin to protein.
In the following assay, the final reaction volume is 150 µl. The buffer in which the protein and F-actin are incubated will depend on the protein to
be tested and on the conditions required. In this example, we use a buffer containing 10 mM Tris pH 7.0, 1 mM ATP, 0.2 mM DTT, 1 mM EGTA,
0.1 mM CaCl2and 2 mM MgCl2. The protein and F-actin are incubated in the buffer for 1 hour at room temperature.
4. Sedimentation of F-actin
Following the incubation, the samples are spun at 100,000g at 22ºC. We use a Beckman ultracentrifuge in the video. The rotor is carefully
removed so as not to disturb the pellets.
5. Analysis of protein co-sedimentation with F-actin
The supernatants are carefully removed by pipetting into an eppendorf tube and 5 X Laemmli SDS-PAGE sample buffer is added. An appropriate
volume of 1 X Laemmli SDS-PAGE sample buffer is added to the pellets remaining, pipetted up and down, and transferred to new eppendorf
tubes. The relative amounts of protein in the pellets and supernatants are analyzed following their separation by SDS-PAGE and Coomassie Blue
staining of the gel or Western blotting.
In order to determine the specificity of protein interaction with actin, actin concentration-dependent co-sedimentations can be performed. For this
sort of experiment, a fixed amount of the protein and a series of increasing amounts of actin are incubated, and analysis is carried out as
Actin co-sedimentation is a simple in vitro assay to analyze specfic proteins binding to F-actin. In this video, we demonstrate one example of how a
simple actin co-sedimentation can be carried out. We show how to prepare F-actin, prepare the protein to be tested and the procedure of actin
co-sedimentation. A number of points should be considered when carrying out actin co-sedimentation assays. For instance, the buffer components
for the incubation can vary depending on the protein to be tested and pH, temperature and salt concentration may affect the binding to F-actin. We
have demonstrated a simple binding to F-actin, however this type of assay can be elaborated upon and used to determine binding specificity of a
protein or protein domain to F-actin.
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Thank you to Dr. Praveen Kumar in the Wittman lab at UCSF for the movie of actin in HaCAT cells.
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Hip12 with actin. Biochemistry. 14;43(49):15418-28 (2004).
2. Goldmann, W.H., Hess, D., Isenberg, G. The effect of intact talin and talin tail fragment on actin filament dynamics and structure depends on pH
and ionic strength. Eur J Biochem. 260(2):439-45 (1999).
3. Lee, H., Bellin, R.M., Walker, D.L., Patel, B., Powers, P., Liu, H., Garcia-Alvarez, B., de Pereda, J., Liddington, R.C., Volkmann, N., Hanein, D.,
Critchley, D.R. and Robson, R.M. Characterization of an Actin-binding Site within the Talin FERM Domain. J Mol Biol. 22;343(3):771-84 (2004).
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