ROCK enzymatic assay

Abstract

We describe the protocols for measuring Rho-associated coiled-coil- containing kinase (ROCK) activity in vitro. A His-tagged, constitutively active form of the protein (lacking C-terminal inhibitory domains) is expressed in baculovirus. The protein is purified by a combination of metal affinity, ion exchange, and size exclusion chromatography. Enzymatic activity is measured spectrophotometrically in a coupled assay format wherein a molecule of NADH is oxidized to NAD+ each time a phosphate is transferred by ROCK. © 2009 Humana Press.