26] Use of actin filament and microtubule affinity chromatography to identify proteins that bind to the cytoskeleton

Abstract

The proteins that bind to actin filaments and microtubules play an important role in determining the structure and function of these filaments in eukaryotic cells. A number of approaches have been used to identify and characterize these proteins. Actin-binding proteins (ABPs) have been identified primarily by virtue of their ability to affect actin polymerization or actin filament motility in vitro. This chapter describes affinity chromatography methods for the isolation of proteins that bind to actin filaments and microtubules. The use of affinity chromatography offers a number of advantages over the use of other procedures for the purification of cytoskeletal proteins. Large extents of purification are obtained without requiring an activity assay. The agarose matrix used for construction of affinity columns consists of a 1:1 mixture of Affi-Gel 10 and Sepharose CL-6B. Affinity columns constructed from stabilized actin filaments and microtubules will contain a large proportion of protein that is not covalently bound to the column matrix, since a filament needs to be linked to the matrix only at scattered sites to remain bound to the column.