Optimization of a nonradioactive immunosorbent assay for p38alpha mitogen-activated protein kinase activity

Here we describe an optimization of a nonradioactive immunosorbent p38alpha mitogen-activated protein kinase (MAPK) activity assay to determine inhibitory potency of small molecule inhibitors. The assay omits the secondary antibody and, therefore, is shorter, more accurate, and easier to handle (total assay time of 3 h). This direct assay uses a new monoclonal anti-phospho-ATF-2 (Thr69/71) peroxidase-conjugated antibody, increasing specificity and eliminating problems with cross-reactivities of secondary antibodies.