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Journal of Biomolecular Screening
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Article

Development of a Microsphere-Based p38{alpha} MAP Kinase No-Wash Assay

Stefan Laufer* Sabine Linsenmaier

Institute of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry, Eberhard-Karls-University of Tübingen, Germany.

* To whom correspondence should be addressed. E-mail: Stefan.Laufer{at}Uni-Tuebingen.De.


  Abstract

A nonradioactive, microsphere-based, no-wash assay for the measurement of p38{alpha} mitogen-activated protein (MAP) kinase activity was established. In this assay, a glutathione-S-transferase activating transcription factor 2 (amino acids 19-96) fusion protein (GST-ATF-2) was used as substrate for p38{alpha} MAP kinase. The assay involves immobilization of GST-ATF-2 on glutathione-microspheres (GSH-microspheres), addition of test solution containing p38{alpha} MAP kinase and test compounds, and measurement of the respective substrate phosphorylation with the aid of a bi-phospho-specific antibody. The optimization of test conditions is described in this article. With an optimized standard protocol, p38{alpha} MAP kinase inhibitors were investigated and IC50 values were compared to those derived using known assays. This assay might be useful in testing drug candidates.

Key Words: microsphere, p38, kinase assay, Luminex, bead

First published on June 7, 2006, doi:10.1177/1087057106288187

Journal of Biomolecular Screening 2006;11:528.

A more recent version of this article appeared on August 1, 2006

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