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Micro-trap phosphorylation assay of mitogen-activated protein (MAP) kinases to detect their activation by
1Department of Biochemistry, Faculty of Medicine, University of Tokyo, Japan.
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.
Insights
A novel microplate assay efficiently purifies and measures mitogen-activated protein (MAP) kinase activity in macrophages. This sensitive, rapid method aids in studying MAP kinase activation and drug inhibition effects.
Area of Science:
- Biochemistry
- Cell Biology
- Assay Development
Background:
- Mitogen-activated protein (MAP) kinases are crucial signaling molecules.
- Existing methods for MAP kinase detection are often time-consuming and less sensitive.
- A need exists for a more efficient and scalable assay for MAP kinase activity.
Purpose of the Study:
- To develop a microplate-based assay for quantifying MAP kinase activity.
- To compare the novel assay's performance against conventional methods.
- To utilize the assay to investigate MAP kinase activation in macrophages.
Main Methods:
- Partial purification of MAP kinases from murine macrophages using anion-exchanger resin in 96-well plates.
- Development of a micro-trap phosphorylation assay for enzyme activity measurement.
- Stimulation of macrophages with lipopolysaccharide (LPS) and treatment with inhibitors.
Main Results:
- The micro-trap assay demonstrated comparable enzyme activity to traditional methods.
- The assay showed high sensitivity, economy, and rapid execution for large sample numbers.
- LPS stimulation persistently increased MAP kinase activity in macrophages.
- Polymyxin B and tyrosine kinase inhibitors effectively inhibited LPS-induced MAP kinase activation.
Conclusions:
- The microplate-based micro-trap phosphorylation assay is a sensitive, economical, and rapid tool for MAP kinase activity measurement.
- This assay facilitates the study of MAP kinase signaling pathways in various biological contexts.
- The method is suitable for high-throughput screening of drug inhibitors and antisense oligonucleotides targeting MAP kinases.