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Journal of Biomolecular Screening
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Development of a Microsphere-Based p38{alpha} Kinase No-Wash Assay

Stefan Laufer

Eberhard-Karls-University of Tübingen, Institute of Pharmacy Department of Pharmaceutical and Medicinal Chemistry, Auf Der Morgenstelle 8 D-72076 Tübingen, Germany Stefan.Laufer{at}Uni-Tuebingen.De

Sabine Linsenmaier

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

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

This version was published on August 1, 2006

Journal of Biomolecular Screening, Vol. 11, No. 5, 528-536 (2006)
DOI: 10.1177/1087057106288187
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