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Methods Enzymol. 2019;626:23-40. doi: 10.1016/bs.mie.2019.07.014. Epub 2019 Aug 13.

Purification and enzymatic assay of class I histone deacetylase enzymes.

Author information

1
Stowers Institute for Medical Research, Kansas City, MO, United States.
2
Stowers Institute for Medical Research, Kansas City, MO, United States; Department of Pathology & Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS, United States. Electronic address: mpw@stowers.org.

Abstract

The reversible acetylation of histones has a profound influence on transcriptional status. Histone acetyltransferases catalyze the addition of these chemical modifications to histone lysine residues. Conversely, histone deacetylases (HDACs) catalyze the removal of these acetyl groups from histone lysine residues. As modulators of transcription, HDACs have found themselves as targets of several FDA-approved chemotherapeutic compounds which aim to inhibit enzyme activity. The ongoing efforts to develop targeted and isoform-specific HDAC inhibitors necessitates tools to study these modifications and the enzymes that maintain an equilibrium of these modifications. In this chapter, we present an optimized workflow for the isolation of recombinant protein and subsequent assay of class I HDAC activity. We demonstrate the application of this assay by assessing the activities of recombinant HDAC1, HDAC2, and SIN3B. This assay system utilizes readily available reagents and can be used to assess the activity and responsiveness of class I HDAC complexes to HDAC inhibitors.

KEYWORDS:

Chromatin; HDAC inhibitor; HDAC1; HDAC2; Vorinostat

PMID:
31606077
PMCID:
PMC6839770
[Available on 2020-08-13]
DOI:
10.1016/bs.mie.2019.07.014

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