Immune-complex assays for tyrosine protein kinases

Current Protocols in Immunology
A L Burkhardt, J B Bolen

Abstract

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.

References

Dec 1, 1992·Proceedings of the National Academy of Sciences of the United States of America·J D SilicianoS V Desiderio
Dec 1, 1992·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·J B BolenA Y Tsygankov
Oct 1, 1992·Proceedings of the National Academy of Sciences of the United States of America·J E HutchcroftJ M Oliver
May 14, 1992·Nature·T J MolinaA Veillette
Aug 15, 1991·Proceedings of the National Academy of Sciences of the United States of America·A L BurkhardtJ J Mond
Jan 1, 1991·Methods in Enzymology·T Hunter
Apr 20, 1990·Cell·A Ullrich, J Schlessinger
Jun 1, 1990·Proceedings of the National Academy of Sciences of the United States of America·L E SamelsonR D Klausner
Jan 15, 1993·Proceedings of the National Academy of Sciences of the United States of America·S D Heyeck, L J Berg

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Citations

Nov 1, 1996·Japanese Journal of Cancer Research : Gann·Y YamashitaH Mano
Mar 29, 2008·PLoS Computational Biology·Sichun Yang, Benoît Roux
Jan 1, 1996·Stem Cells·K E Yates, J C Gasson

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