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Wnt Signaling pp 197-205 | Cite as

ROCK Enzymatic Assay

  • John D. Doran
  • Marc D. Jacobs
Part of the Methods in Molecular Biology™ book series (MIMB, volume 468)

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.

Key words

Kinase Phosphorylation Coupled-assay Baculovirus Dimer Rho ROCK 

Notes

Acknowledgments

We thank Stephen Chambers, Douglas Austen, Scott Raybuck, and Edward Fox for helpful discussions and critical comments on this manuscript.

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Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • John D. Doran
    • 1
  • Marc D. Jacobs
    • 2
  1. 1.Protein BiochemistryVertex PharmaceuticalsCambridgeUSA
  2. 2.Structural BiologyVertex PharmaceuticalsCambridgeUSA

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