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.
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