Encyclopedia of Systems Biology

2013 Edition
| Editors: Werner Dubitzky, Olaf Wolkenhauer, Kwang-Hyun Cho, Hiroki Yokota

Protein Kinase Assay

  • Joseph R. Pomerening
Reference work entry
DOI: https://doi.org/10.1007/978-1-4419-9863-7_1330



A technique used to measure the activity of a protein phospho-transferase (kinase) that is generally performed in vitro with purified components (e.g., by immunoprecipitation or other purification method), but can also be performed using a diluted cell lysate (e.g., a Xenopus egg extract) if the affinity of the enzyme for the substrate is significant. While non-radioactive systems have been developed, a majority of investigators use radioactive assays because of increased sensitivity and stability of the labeled reaction product. In addition to the presence of magnesium in these reactions (kinases are magnesium dependent), radioactive protein kinase assays contain higher concentrations of unlabeled adenosine triphosphate (ATP) – sufficient to exceed the Km of the enzyme – and a low concentration of isotopically labeled ATP (the γ-phosphate is either 32P or 33P) to permit the detection of substrate by autoradiographic methods....

This is a preview of subscription content, log in to check access.


  1. Beaudette KN, Lew J, Wang JH (1993) Substrate specificity characterization of a cdc2-like protein kinase purified from bovine brain. J Biol Chem 268(28):20825–20830PubMedGoogle Scholar
  2. Hastie CJ, McLauchlan HJ, Cohen P (2006) Assay of protein kinases using radiolabeled ATP: a protocol. Nat Protoc 1(2):968–971PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  1. 1.Department of BiologyIndiana UniversityBloomingtonUSA