Actin co-sedimentation assay; for the analysis of protein binding to F-actin

Department of Cell and Tissue Biology, University of California, San Francisco, USA.
Journal of Visualized Experiments (Impact Factor: 1.33). 02/2008; 28pii(13). DOI: 10.3791/690
Source: PubMed


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 assays.

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    • "After incubation, samples were centrifuged at 55,000 rpm at 22 • C for 1 h, and supernatants were carefully removed. Supernatants and pellets were then treated in 5× and 1× Laemmli SDS–PAGE sample, respectively, and analyzed by SDS–PAGE and Coomassie Blue staining of the gel [24]. AGS cells were cultured to about 50–70% confluence in six-well plates, harvested, and transfected by electroporation in a Bio-Rad gene pulser (about 5 × 10 6 cells/sample at 1300 V, 10 ms for 3 times). "
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