Development of a microsphere-based p38alpha MAP kinase no-wash assay.
A nonradioactive, microsphere-based, no-wash assay for the measurement of p38alpha 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 p38alpha MAP kinase. The assay involves immobilization of GST-ATF-2 on glutathione-microspheres (GSH-microspheres), addition of test solution containing p38alpha 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, p38alpha 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.
Laufer
Stefan
S
Eberhard-Karls-University of Tübingen, Institute of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry, Auf Der Morgenstelle 8 D-72076 Tübingen, Germany. Stefan.Laufer@Uni-Tuebingen.De
Linsenmaier
Sabine
S
D051698:Activating Transcription Factor 2 / Q000276:immunology / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001667:Binding, Competitive; D004121:Dimethyl Sulfoxide / Q000009:adverse effects; D004791:Enzyme Inhibitors / Q000378:metabolism; D005456:Fluorescent Dyes / Q000032:analysis; D005982:Glutathione Transferase / Q000378:metabolism; D007124:Immunoenzyme Techniques / Q000379:methods*; D020128:Inhibitory Concentration 50; D008863:Microspheres; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*
D051698:Activating Transcription Factor 2; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D005982:Glutathione Transferase; D048051:p38 Mitogen-Activated Protein Kinases; D004121:Dimethyl Sulfoxide