Array-based fluorescence assay for serine/threonine kinases using specific chemical reaction
- Bioorg Med Chem. 2008 Aug 15;16(16):7788-94. doi: 10.1016/j.bmc.2008.07.007.
- 1. Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan.
We report herein the development of an efficient fluorescence assay for serine/threonine kinases using a peptide array. Our approach is based on chemical reactions specific to phosphoserine and phosphothreonine residues, that is, base-mediated beta-elimination of the phosphate group and subsequent Michael addition of a thiol-containing fluorescent reagent. This procedure enables the covalent introduction of a fluorescent moiety into the phosphorylated peptide. Novel fluorescent reagents were designed for this purpose and synthesized. With these reagents, protein kinase A (PKA) and Akt-1 activities were readily detected. Our method can also be used to measure the activity of kinase inhibitors. This assay is expected to be widely applicable in kinase research.