title: High-throughput kinase assay based on surface plasmon resonance.
abstract: We have designed a novel high-throughput (HTP) kinase assay using an array-based surface plasmon resonance (SPR) apparatus. For high flexibility and performance, the kinase assay procedure is divided into an in vitro phosphorylation part and a phospho-detection part on a sensor chip. Not only biotinylated peptides but also recombinant proteins fused with FLAG-GST tandem tag can be used as native substrates. The substrate is selectively captured by a capture antibody immobilized on a sensor chip, and phospho-tyrosine (pTyr) residues are detected by an anti-pTyr antibody. The level of tyrosine phosphorylation is calculated from the capture level of the substrates and the binding level of the anti-pTyr antibody monitored by SPR. A wide dynamic range and real-time monitoring of SPR contribute to improved data reliability, and optimization of the procedure for an array-based apparatus achieved multiple sample processing (1,000 samples/day).
authors.affiliation: Protein Expression Team, Japan Biological Information Research Center, Japan Biological Informatics Consortium, Tokyo, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Takeda
authors.forename: Naoki
authors.initials: N
authors.lastname: Goshima
authors.forename: Nobuo
authors.initials: N
authors.lastname: Nomura
mesh_terms: D000906:Antibodies / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D057075:Enzyme Assays / Q000295:instrumentation / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000276:immunology / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D020349:Surface Plasmon Resonance / Q000295:instrumentation / Q000379:methods*
chemical_list: D000906:Antibodies; D055767:Immobilized Proteins; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases