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

25 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

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

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title = "A non-isotopic in vitro assay for histone acetylation",

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.",

keywords = "Acetyl-transferase assay, Histone H3, Histone H4, Non-isotopic, PCAF, p300",

author = "David Kuninger and James Lundblad and Anthony Semirale and Peter Rotwein",

note = "Funding Information: 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.",

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T1 - A non-isotopic in vitro assay for histone acetylation

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

Keywords

ASJC Scopus subject areas

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