The fluorescence-based acetylation assay using thiol-sensitive probes

Tielong Gao, Chao Yang, Yujun George Zheng

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Lysine acetyltransferases (KATs) catalyze the acetylation of specific lysine residues in histone and nonhistone proteins. The enzymatic activities of KATs are involved in a broad spectrum of cellular processes. Thus far, the reaction of KAT catalysis has been studied by various bioanalytical methods such as radioisotopic labeling, spectrophotometric and fluorometric measurements, and antibody-dependent immunosorbent assays. In particular, the fluorescent method has the advantage of simplicity for implementation, fast assay speed, fine signal to noise ratio, and superior sensitivity. We describe here the technical protocols of using thiolsensitive fluorogenic probes for the fluorescent analysis of enzymatic activities of KATs, with males on the first (MOF) as an exemplary KAT enzyme. 7-Diethylamino-3-(4'-maleimidylphenyl)-4- methylcoumarin (CPM) is selected as the KAT probe owing to its fast reaction kinetics with coenzyme A (CoA) and excellent fluorogenicity upon thiol conjugation. The fluorescence-based acetylation assay is well suited for both kinetic characterization of KAT catalysis and KAT inhibitor investigation.

Original languageEnglish (US)
Title of host publicationProtein Acetylation
Subtitle of host publicationMethods and Protocols
PublisherHumana Press Inc.
Pages229-238
Number of pages10
ISBN (Print)9781627033046
DOIs
StatePublished - 2013
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume981
ISSN (Print)1064-3745

Keywords

  • Acetylation
  • Fluorescence
  • HAT
  • Histone
  • KAT
  • Lysine acetyltransferase
  • MYST

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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