Non-isotopic in vitro assay for histone acetylation
Non-isotopic in vitro assay for histone acetylation
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DOI：
10.1016/j.jbiotec.2007.07.498
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发表时间：
2007-09-15
影响因子：
4.1
通讯作者：
Rotwein, Peter
中科院分区：
文献类型：
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作者：
Kuninger, David;Lundblad, James;Rotwein, Peter
我们描述了一种简单，稳健，相对便宜的非放射性体外测定组蛋白乙酰转移酶活性。该方法利用了重组大肠杆菌易于纯化的优点。大肠杆菌衍生的融合蛋白，其含有与表位标记的麦芽糖结合蛋白（MBP）连接的组蛋白H3和H4的NH 2末端尾部，并用对乙酰化的H3和H4特异的抗体进行免疫印迹。在这里，我们显示了特异性和动态范围的组蛋白乙酰转移酶，p300和PCAF的测定。该测定法可以通过简单地产生新的融合蛋白而容易地适用于其他底物，并且通过修改反应条件而适用于其他乙酰基转移酶。(C)2007 Elsevier B.V.保留所有权利。
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.