A non-isotopic in vitro assay for histone acetylation

David Kuninger; James Lundblad; Anthony Semirale; Peter Rotwein

doi:10.1016/j.jbiotec.2007.07.498

A non-isotopic in vitro assay for histone acetylation

David Kuninger
, James Lundblad
, Anthony Semirale
, Peter Rotwein

Research output: Contribution to journal › Article › peer-review

26 Scopus citations

Abstract

We describe a simple, robust, and relatively inexpensive non-radioactive in vitro assay for measuring histone acetyl-transferase activity. The assay takes advantage of easy to purify recombinant E. coli-derived fusion proteins containing the NH₂-terminal tails of histones H3 and H4 linked to epitope-tagged maltose-binding protein (MBP), and immunoblotting with antibodies specific to acetylated H3 and H4. Here we show the specificity and dynamic range of this assay for the histone acetyl-transferases, p300 and PCAF. This assay may be adapted readily for other substrates by simply generating new fusion proteins and for other acetyl-transferases by modifying reaction conditions.

Original language	English (US)
Pages (from-to)	253-260
Number of pages	8
Journal	Journal of Biotechnology
Volume	131
Issue number	3
DOIs	https://doi.org/10.1016/j.jbiotec.2007.07.498
State	Published - Sep 15 2007
Externally published	Yes

Funding

These studies were supported by NIH Grants RO1 DK42748 and RO1 DK63073 (to P.R.). We thank Ryan Kuzmickas for technical assistance during the early phases of this project.

Funders	Funder number
Author National Institutes of Health National Institutes of Health National Institutes of Health National Institutes of Health The Bev Hartig Huntington's Disease Foundation National Institutes of Health	RO1 DK63073, RO1 DK42748
National Institute of Diabetes and Digestive and Kidney Diseases	T32DK007674

Keywords

Acetyl-transferase assay
Histone H3
Histone H4
Non-isotopic
PCAF
p300

ASJC Scopus subject areas

Biotechnology
Bioengineering
Applied Microbiology and Biotechnology

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10.1016/j.jbiotec.2007.07.498

Cite this

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abstract = "We describe a simple, robust, and relatively inexpensive non-radioactive in vitro assay for measuring histone acetyl-transferase activity. The assay takes advantage of easy to purify recombinant E. coli-derived fusion proteins containing the NH2-terminal tails of histones H3 and H4 linked to epitope-tagged maltose-binding protein (MBP), and immunoblotting with antibodies specific to acetylated H3 and H4. Here we show the specificity and dynamic range of this assay for the histone acetyl-transferases, p300 and PCAF. This assay may be adapted readily for other substrates by simply generating new fusion proteins and for other acetyl-transferases by modifying reaction conditions.",

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AU - Kuninger, David

AU - Lundblad, James

AU - Semirale, Anthony

AU - Rotwein, Peter

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AB - We describe a simple, robust, and relatively inexpensive non-radioactive in vitro assay for measuring histone acetyl-transferase activity. The assay takes advantage of easy to purify recombinant E. coli-derived fusion proteins containing the NH2-terminal tails of histones H3 and H4 linked to epitope-tagged maltose-binding protein (MBP), and immunoblotting with antibodies specific to acetylated H3 and H4. Here we show the specificity and dynamic range of this assay for the histone acetyl-transferases, p300 and PCAF. This assay may be adapted readily for other substrates by simply generating new fusion proteins and for other acetyl-transferases by modifying reaction conditions.

KW - Acetyl-transferase assay

KW - Histone H3

KW - Histone H4

KW - Non-isotopic

KW - PCAF

KW - p300

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A non-isotopic in vitro assay for histone acetylation

Abstract

Funding

Keywords

ASJC Scopus subject areas

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