Non-isotopic in vitro assay for histone acetylation

被引：23
作者：
Kuninger, David
Lundblad, James
Semirale, Anthony
Rotwein, Peter
机构：
[1] Oregon Hlth & Sci Univ, Dept Biochem & Mol Biol, Portland, OR 97239 USA
[2] Oregon Hlth & Sci Univ, Dept Med, Portland, OR 97239 USA
来源：
JOURNAL OF BIOTECHNOLOGY | 2007年 / 131卷 / 03期
关键词：
non-isotopic; acetyl-transferase assay; p300; PCAF; histone h3; histone h4;
D O I：
10.1016/j.jbiotec.2007.07.498
中图分类号：
Q81 [生物工程学（生物技术）]; Q93 [微生物学];
学科分类号：
071005 ; 0836 ; 090102 ; 100705 ;
摘要：
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. (C) 2007 Elsevier B.V. All rights reserved.
引用
收藏
页码：253 / 260
页数：8
相关论文
共 22 条
[1]   A rapid and sensitive assay for histone acetyl-transferase activity [J].
Ait-Si-Ali, S ;
Ramirez, S ;
Robin, P ;
Trouche, D ;
Harel-Bellan, A .
NUCLEIC ACIDS RESEARCH, 1998, 26 (16) :3869-3870
[2]   The transcriptional regulatory code of eukaryotic cells insights from genome-wide analysis of chromatin organization and transcription factor binding [J].
Barrera, Leah O. ;
Ren, Bing .
CURRENT OPINION IN CELL BIOLOGY, 2006, 18 (03) :291-298
[3]  
BEMDSEN CE, 2005, METHODS, V36, P321
[4]   The histone acetyltransferase activity of human GCN5 and PCAF is stabilized by coenzymes [J].
Herrera, JE ;
Bergel, M ;
Yang, XJ ;
Nakatani, Y ;
Bustin, M .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1997, 272 (43) :27253-27258
[5]   CBP and p300: HATs for different occasions [J].
Kalkhoven, E .
BIOCHEMICAL PHARMACOLOGY, 2004, 68 (06) :1145-1155
[6]   A continuous, nonradioactive assay for histone acetyltransferases [J].
Kim, Y ;
Tanner, KG ;
Denu, JM .
ANALYTICAL BIOCHEMISTRY, 2000, 280 (02) :308-314
[7]  
Kraus WL, 1999, MOL CELL BIOL, V19, P8123
[8]   Muscle cell survival mediated by the transcriptional coactivators p300 and PCAF displays different requirements for acetyltransferase activity [J].
Kuninger, David ;
Wright, Alistair ;
Rotwein, Peter .
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY, 2006, 291 (04) :C699-C709
[9]   The role of chromatin during transcription [J].
Li, Bing ;
Carey, Michael ;
Workman, Jerry L. .
CELL, 2007, 128 (04) :707-719
[10]  
Mizzen CA, 1999, METHOD ENZYMOL, V304, P675
123