Aim: To develop a homogenous, nonradioactive, antibody-free and universal assay for diverse families of methyltransferases and monitor the activity of these enzymes in a high-throughput format.
Materials & Methods: The assay conditions are optimized for monitoring the enzymatic activity of a broad range of methyltransferases regardless of the chemical structure or nature of the enzyme substrate in a low- and high-throughput-formatted protocols. The assay detects S-adenosyl-L-homocysteine, the universal reaction products of all methyltransferases.
Results: We demonstrate the utility of using this protocol to determine the activity of DNA, protein methyltransferases and also to determine kinetic parameters of several inhibitors using purified enzymes. The assay is sensitive (20-30 nM of S-adenosyl-L-homocysteine) and robust.
Conclusion: The methyltransferase Glo is nonradioactive, antibody-free and homogenous, universal assay to determine enzyme activity of diverse families of methyltransferases. The assay is formatted to meet the requirements of high-throughput screening in drug discovery programs searching for modulators of methyltransferases.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.2217/epi.15.113 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
New strategy to address DNA-methyl transferase activity in ovarian cancer cell cultures by monitoring the formation of 5-methylcytosine using HPLC-UV.
J Chromatogr B Analyt Technol Biomed Life Sci
August 2016
Department of Physical and Analytical Chemistry, Faculty of Chemistry, University of Oviedo, C/Julian Clavería 8, 33006 Oviedo, Spain. Electronic address:
Methylation of mammalian genomic DNA is catalyzed by DNA methyltransferases (DNMTs). Aberrant expression and activity of these enzymes has been reported to play an important role in the initiation and progression of tumors and its response to chemotherapy. Therefore, there is a great interest in developing strategies to detect human DNMTs activity.
View Article and Find Full Text PDFMethyltransferase-Glo: a universal, bioluminescent and homogenous assay for monitoring all classes of methyltransferases.
Epigenomics
March 2016
Promega Corporation, R&D Department, 2800 Woods Hollow Road, Madison, WI 53711, USA.
Aim: To develop a homogenous, nonradioactive, antibody-free and universal assay for diverse families of methyltransferases and monitor the activity of these enzymes in a high-throughput format.
Materials & Methods: The assay conditions are optimized for monitoring the enzymatic activity of a broad range of methyltransferases regardless of the chemical structure or nature of the enzyme substrate in a low- and high-throughput-formatted protocols. The assay detects S-adenosyl-L-homocysteine, the universal reaction products of all methyltransferases.
Strategies to detect human DNA methyltransferases are needed, given that aberrant methylation by these enzymes is associated with cancer initiation and progression. Here we describe a nonradioactive, antibody-free, electrochemical assay in which methyltransferase activity on DNA-modified electrodes confers protection from restriction for signal-on detection. We implement this assay with a multiplexed chip platform and show robust detection of both bacterial (SssI) and human (Dnmt1) methyltransferase activity.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!