Continuous enzymatic assay for histone lysine methyltransferases. (November 2007)

• Record Type: Journal Article
• Authors: Rathert, Philipp; Cheng, Xiaodong; Jeltsch, Albert
• Is Part Of: Biotechniques. Volume 43:Number 5(2007)
• Page Start: 602
• Page End: 608
• Publication Date: 2007-11
• Subjects: Biology, Experimental -- Periodicals; Molecular biology -- Periodicals; Medical technology -- Periodicals; Biology, Experimental; Medical technology; Molecular biology; Clinical Laboratory Techniques -- Periodicals; Research -- Periodicals; Medical Laboratory Science -- Periodicals; Periodicals; Electronic journals; 570
• DOI: 10.2144/000112623
• Languages: English
• ISSNs: 0736-6205

Abstract

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 suited for 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.