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J Vis Exp. 2008 Mar 28;(13). pii: 690. doi: 10.3791/690.

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

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  • 1Department of Cell and Tissue Biology, University of California, San Francisco, USA.

Abstract

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.

PMID:
19066573
[PubMed - indexed for MEDLINE]
PMCID:
PMC2586866
Free PMC Article
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