UNIT 11.4 Immune-Complex Assays for Tyrosine Protein Kinases

  1. Anne L. Burkhardt,
  2. Joseph B. Bolen

Published Online: 1 MAY 2001

DOI: 10.1002/0471142735.im1104s07

Current Protocols in Immunology

Current Protocols in Immunology

How to Cite

Burkhardt, A. L. and Bolen, J. B. 2001. Immune-Complex Assays for Tyrosine Protein Kinases. Current Protocols in Immunology. 7:11.4:11.4.1–11.4.9.

Author Information

  1. Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: SEP 1993


Tyrosine protein kinases (TPKs) represent a diverse group of enzymes that contribute to cellular signal transduction. The generally low abundance of TPKs, coupled with their rapid activation and deactivation, usually precludes their purification through conventional biochemical means. Using immune-complex protein kinase assays, the presence or absence of a given TPK can be established and an estimation of its functional state obtained. In the Basic Protocol of this unit, TPKs are immunoprecipitated, allowed to autophosphorylate in the presence of labeled ATP, run out on an SDS-PAGE gel, and detected by autoradiography. Alternate protocols are provided for the assessment of the functional state of TPKs by providing a potential substrate along with the labeled ATP in the reaction mixture. In the first alternate protocol, the exogenous substrate is a protein, permitting simultaneous assessment of autophosphorylation and exogenous substrate phosphorylation. The second alternate protocol utilizes a peptide substrate, resulting in a rapid, high-throughput assay that evaluates only exogenous substrate phosphorylation.