High-throughput kinase assay based on surface plasmon resonance.

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).