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.
Brown
Jeffrey W
JW
Department of Physiology and Biophysics, Boston University School of Medicine, 700 Albany Street, Boston, MA 02118, USA.
McKnight
C James
CJ
D000199:Actins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D008840:Microfilament Proteins / Q000032:analysis; D013056:Spectrophotometry, Ultraviolet
D000199:Actins; D008840:Microfilament Proteins; C026615:villin