MICRO-TRAP PHOSPHORYLATION ASSAY OF MITOGEN-ACTIVATED PROTEIN (MAP) KINASES TO DETECT THEIR ACTIVATION BY LIPOPOLYSACCHARIDES

We designed a microplate-based assay method for mitogen-activated prot ein (MAP) kinase. Using anion-exchanger resin, MAP kinases from murine macrophages were partially purified in 96-well plates. The activities of these purified enzymes correlated well with those detected in here tofore used assays. The micro-trap phosphorylation assay has advantage s over conventional methods (immunoprecipitation, Western blotting for the detection of mobility shift, or kinase detection assay in myelin basic protein (MBP)-containing gel), in terms of sensitivity, economy and rapid execution for hundreds of samples. Using micro-trap phosphor ylation assay, It was demonstrated that MAP kinase activities in macro phages were persistently increased by lipopolysaccharide (LPS) stimula tion, and this activation was inhibited by polymyxin B or tyrosine kin ase inhibitors. This method is expected to give a wide range of applic ation, such as determining effects of drug inhibitors or antisense oli gonucleotides on MAP kinases, or measuring the various protein kinases after specificity controls were done.