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 protein (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 heretofore used assays. The micro-trap phosphorylation assay has advantages 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 phosphorylation assay, it was demonstrated that MAP kinase activities in macrophages were persistently increased by lipopolysaccharide (LPS) stimulation, and this activation was inhibited by polymyxin B or tyrosine kinase inhibitors. This method is expected to give a wide range of application, such as determining effects of drug inhibitors or antisense oligonucleotides on MAP kinases, or measuring the various protein kinases after specificity controls were done.
Waga
I
I
Department of Biochemistry, Faculty of Medicine, University of Tokyo, Japan.
Kume
K
K
Ferby
I
I
Honda
Z
Z
Shimizu
T
T
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000032:analysis* / Q000378:metabolism; D002460:Cell Line; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000201:enzymology; D051379:Mice; D010766:Phosphorylation; D011112:Polymyxin B / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors
D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D011505:Protein-Tyrosine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D011112:Polymyxin B