Title |
Development of a high-throughput fluoroimmunoassay for Syk kinase and Syk kinase inhibitors
|
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Published in |
Analytical Biochemistry, April 2003
|
DOI | 10.1016/s0003-2697(03)00026-5 |
Pubmed ID | |
Authors |
Noriyuki Yamamoto, Haruki Hasegawa, Hitomi Seki, Karl Ziegelbauer, Takahiro Yasuda |
Abstract |
Syk is a tyrosine kinase which is indispensable in immunoglobulin Fc receptor- and B cell receptor-mediated signal transduction in various immune cells. This pathway is important in the pathophysiology of allergy. In this study we established a quantitative nonradioactive kinase assay to identify inhibitors of Syk. We used recombinant GST-tagged Syk purified from baculovirus-infected insect cells. As a substrate, biotinylated peptide corresponding to the activation loop domain of Syk, whose tyrosine residues are autophosphorylated upon activation, was employed to screen both ATP- and substrate-competitive inhibitors. After the kinase reaction in solution phase, substrate was trapped on a streptavidin-coated plate, followed by detection of the phosphorylated tyrosine with europium-labeled anti-phosphotyrosine antibody. The kinase reaction in solution phase greatly enhanced phosphorylation of substrate compared to that of plate-coated substrate. High signal-to-background ratio and low data scattering were obtained in the optimized high-throughput screening (HTS) format. Further, several kinase inhibitors showed concentration-dependent inhibition of recombinant Syk kinase activity with almost the same efficacy for immunoprecipitated Syk from a human cell line. These data suggest that this assay is useful to screen Syk kinase inhibitors in HTS. |
Mendeley readers
Geographical breakdown
Country | Count | As % |
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Unknown | 12 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
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Researcher | 5 | 42% |
Student > Master | 2 | 17% |
Other | 1 | 8% |
Lecturer > Senior Lecturer | 1 | 8% |
Student > Ph. D. Student | 1 | 8% |
Other | 0 | 0% |
Unknown | 2 | 17% |
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Pharmacology, Toxicology and Pharmaceutical Science | 1 | 8% |
Other | 0 | 0% |
Unknown | 1 | 8% |