Micro-trap phosphorylation assay of mitogen-activated protein (MAP) kinases to detect their activation by lipopolysaccharides
| Autor: | Ingvar Ferby, Iwao Waga, Takao Shimizu, Kazuhiko Kume, Zen-ichiro Honda |
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| Rok vydání: | 1996 |
| Předmět: |
Lipopolysaccharides
Calcium-Calmodulin-Dependent Protein Kinases Kinase Macrophages Immunology Protein-Tyrosine Kinases Mitogen-activated protein kinase kinase Biology Molecular biology Cell Line MAP2K7 Enzyme Activation Mice Biochemistry Mitogen-activated protein kinase biology.protein Animals Immunology and Allergy Phosphorylation Enzyme Inhibitors Tyrosine kinase Polymyxin B MAPK14 |
| Zdroj: | Journal of Immunological Methods. 190:71-77 |
| ISSN: | 0022-1759 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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