Nonradioactive assay of FLAG-tagged MAPK using ANTI-FLAG antibody-coated multiwell plates

We have developed a rapid, sensitive, and quantitative 96-well microplate-based nonradioactive immunoprecipitation/kinase assay to evaluate mitogen-activated protein kinase (MAPK) activity. Three quantitative nonradioactive imunoprecipitation/kinase assays of MAPK were demonstrated on a 96-well microplate coated with ANTI-FLAG M2 antibody (ANTI-FLAG M2 plate): (i) the capture of phosphorylated FLAG-tagged MAPK fusion protein (FLAG-MAPK) from phorbol esters-stimulated, FLAG-MAPK-transfected COS-7 cells, coupled with a very sensitive ELISA procedure to quantitate the level of phosphorylation of FLAG-MAPK; (ii) the in vitro kinase reaction of FLAG-MAPK activity with a substrate and ATP in the same well used to captured the phosphorylated FLAG-MAPK; and (iii) the in vitro kinase reaction of captured non-activated FLAG-MAPK by its upstream kinase from phorbol 12-myristate 13-acetate (PMA)-stimulated COS-7 cells. These results demonstrate that the ANTI-FLAG M2 plate allows for the rapid and quantitative determination of phosphorylation of FLAG-MAPK directly from stimulated, transfected cell lysate. Captured, phosphorylated FLAG-MAPK retains catalytic activity as demonstrated by the phosphorylation of Elk-1 in the same well. Furthermore, phosphorylation of captured FLAG-MAPK by the upstream kinases can be observed directly on the plate. These assays are sensitive, specific, and suitable for handling multiple samples. Thus, the ANTI-FLAG M2 plate forms the basis of a high-throughput screening platform in kinase analysis.