A homogeneous fluorescence polarization assay adaptable for a range of protein serine/threonine and tyrosine kinases.
Recently, a new technology for high-throughput screening has been developed, called IMAP(patent pending). IMAP technology has previously been implemented in an assay for cyclic nucleotide phosphodiesterases (PDE). The authors describe the development of a homogeneous, non-antibody-based fluorescence polarization (FP) assay for a variety of protein kinases. In this assay, fluorescently labeled peptide substrate phosphorylated by the kinase is captured on modified nanoparticles through interactions with immobilized metal (M(III)) coordination complexes, resulting in a change from low to high polarization values. This assay is applicable to protein kinases that phosphorylate serine, threonine, or tyrosine residues. The IMAP platform is very compatible with high-throughput robotics and can be applied to the 1536-well format. As there are hundreds of different kinases coded for in the human genome, the assay platform described in this report is a valuable new tool in drug discovery.
Gaudet
Elizabeth A
EA
Molecular Devices Corporation, 1311 Orleans Ave. Sunnydale, CA 94089, USA. Liz_Gaudet@MolDev.com
Huang
Kuo-Sen
KS
Zhang
Yan
Y
Huang
Wei
W
Mark
David
D
Sportsman
J Richard
JR
D001681:Biological Assay / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000378:metabolism; D005454:Fluorescence Polarization / Q000379:methods*; D006801:Humans; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D019311:Staurosporine / Q000378:metabolism
D004791:Enzyme Inhibitors; D020794:Receptor Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D019311:Staurosporine