Continuous enzymatic assay for histone lysine methyltransferases

被引:27
|
作者
Rathert, Philipp [1 ]
Cheng, Xiaodong
Jeltsch, Albert
机构
[1] Jacobs Univ Bremen, Bremen, Germany
[2] Emory Univ, Sch Med, Atlanta, GA USA
关键词
D O I
10.2144/000112623
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
We describe a continuous peptide methylation assay using the Neurospora crassa Dim-5 histone H3 lysine 9 (H3K9) methyltransferase as a model system. The assay uses streptavidin FlashPlates coated with target peptide. Since no washing and pipeting steps were required after the addition of the enzyme/S-adenosyl-L-methionine (AdoMet) mixture to the microplate, a continuous readout of the reaction progress was possible. We show that this assay is highly reproducible (with errors in the order of +/- 3%). The continuous assay is well suitedfor the simultaneous analysis of up to 384 samples, thus allowing for a rapid screening of methylation rates of different substrates under different conditions or in the presence of inhibitors.
引用
收藏
页码:602 / +
页数:4
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