Identifying competitive protein antagonists for F-actin with reverse-phase high-performance liquid chromatography.

F-actin binding constants are traditionally determined by centrifugal cosedimentation with actin microfilaments, where bound protein is separated from actin with SDS-PAGE and quantitated using densitometry. Here, we demonstrate that UV quantitation of reverse-phase HPLC-separated proteins provides increased accuracy and sensitivity, can be fully automated, and allows one to perform F-actin competition assays on similar sized proteins.