title: High-throughput kinase assay based on surface plasmon resonance suitable for native protein substrates.
abstract: We report a novel in vitro high-throughput (HTP) kinase assay using surface plasmon resonance (SPR). In vitro tyrosine phosphorylation was performed in a microtiter plate, after which the substrate was captured with an antibody on a sensor chip and phosphotyrosine (pTyr) was detected with an anti-pTyr antibody. The capture and pTyr detection steps were performed using a Biacore A100, which is a sensitive and high-performance flow-cell-based SPR biosensor. This system allowed multiple sample processing (1000 samples/day) and high-quality data sampling. We compared the abilities of the HTP-SPR method and a standard radioisotope assay by measuring the phosphorylation of several substrate proteins by the Fyn tyrosine kinase. Similar results were obtained with both methods, suggesting that the HTP-SPR method is reliable. Therefore, the HTP-SPR method described in this study can be a powerful tool for a variety of screening analyses, such as kinase activity screening, kinase substrate profiling, and kinase HTP screening of kinase inhibitors.
authors.affiliation: Protein Expression Team, Japan Biological Information Research Center, Japan Biological Informatics Consortium, Tokyo 135-0064, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Takeda
authors.forename: Akiko
authors.initials: A
authors.lastname: Fukumoto
authors.forename: Aya
authors.initials: A
authors.lastname: Miura
authors.forename: Naoki
authors.initials: N
authors.lastname: Goshima
authors.forename: Nobuo
authors.initials: N
authors.lastname: Nomura
mesh_terms: D000906:Antibodies; D007118:Immunoassay; D010766:Phosphorylation; D010770:Phosphotransferases / Q000032:analysis* / Q000378:metabolism; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance / Q000379:methods*
chemical_list: D000906:Antibodies; D010770:Phosphotransferases