PMID: 8601713Mar 28, 1996Paper

Micro-trap phosphorylation assay of mitogen-activated protein (MAP) kinases to detect their activation by lipopolysaccharides

Journal of Immunological Methods
I WagaT Shimizu

Abstract

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.

References

Oct 1, 1976·Immunochemistry·D C Morrison, D M Jacobs
Jul 15, 1992·Proceedings of the National Academy of Sciences of the United States of America·W H Biggs, S L Zipursky
Dec 15, 1993·Biochemical and Biophysical Research Communications·I WagaT Shimizu
Nov 1, 1993·Trends in Genetics : TIG·A M Neiman
Apr 1, 1993·Trends in Biochemical Sciences·E Nishida, Y Gotoh
Jul 1, 1993·Proceedings of the National Academy of Sciences of the United States of America·J Blenis
Jan 1, 1993·The Plant Cell·B DuerrT Jacobs

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Citations

Jul 11, 2000·Genes to Cells : Devoted to Molecular & Cellular Mechanisms·M AiharaT Shimizu
Jun 9, 1997·Biochemical and Biophysical Research Communications·H EndohH Kawashima
Feb 1, 1997·The American Journal of Physiology·H KatagiriY Oka

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