title: 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.
authors.affiliation: Protein Biochemistry, Vertex Pharmaceuticals, Cambridge, MA, USA.
authors.forename: John D
authors.initials: JD
authors.lastname: Doran
authors.forename: Marc D
authors.initials: MD
authors.lastname: Jacobs
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D054460:rho-Associated Kinases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D054460:rho-Associated Kinases