Array-based fluorescence assay for serine/threonine kinases using specific chemical reaction

Shoji Akita, Naoki Umezawa, Nobuki Kato, Tsunehiko Higuchi

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

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 β-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.

Original languageEnglish
Pages (from-to)7788-7794
Number of pages7
JournalBioorganic and Medicinal Chemistry
Volume16
Issue number16
DOIs
Publication statusPublished - 2008 Aug 15
Externally publishedYes

Keywords

  • Fluorescence spectroscopy
  • High-throughput screening
  • Michael addition
  • Phosphopeptides

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmaceutical Science
  • Drug Discovery
  • Clinical Biochemistry
  • Organic Chemistry

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