The EpiQuik™ DNMT (DNA Methyltransferase) Activity/Inhibition Assay Ultra Kit (Fluorometric) is a complete set of optimized buffers and reagents that allows the experimenter to fluorometrically measure DNA methyltransferase activity or inhibition at extremely fast speeds on a 96-stripwell microplate. The kit is ready-to-use and provides all the essential components needed to carry out a successful DNMT activity/inhibition experiment without the need for radioactivity or any special equipment. The EpiQuik DNMT Activity/Inhibition Assay Ultra Kit is a further refinement of its popular predecessor kit by enhancing sample signals and significantly minimizing background signals, in addition to being ten times more sensitive. The kit has the following advantages and features:
The EpiQuik Nuclear Extraction Kit has also been optimized for use with this kit.
About DNA MethyltransferasesThe addition of methyl groups is carried out by a family of enzymes, DNA methyltransferases (DNMTs or DNA MTases). Chromatin structure in the vicinity of gene promoters also affects DNA methylation and transcriptional activity. DNMT1, DNMT3A, and DNMT3B are required for the establishment and maintenance of DNA methylation patterns. Two additional enzymes, DNMT2/TRDMT1 and DNMT3L, may also have more specialized but related functions. DNMT1 appears to be responsible for maintenance of established patterns of DNA methylation, while DNMT3A and 3B seem to mediate establishment of new or de novo, DNA methylation patterns. DNMT2/TRDMT1 was shown to methylate tRNAAsp at C38 and DNMT3L is found to be a catalytically inactive regulatory factor of DNA methyltransferases, which is essential for the function of DNMT3A and DNMT3B. Diseased cells such as cancer cells may be different in that DNMT1 alone is not responsible for maintaining abnormal gene hypermethylation and both DNMT1 and DNMT3B may cooperate with this function.
Fig. 2. EpiQuik DNMT activity and inhibition fluorometric assay. High sensitivity and specificity achieved by using recombinant DNMT1 with the EpiQuik DNMT Activity/Inhibition Assay Ultra Kit (Fluorometric).
Principle & ProcedureIn the non-radioactive assay with this kit, a universal DNMT substrate is stably coated onto the wells. DNMT enzymes transfer methyl groups to cytosines from Adomet to methylate the DNA substrate. The methylated DNA can be recognized with an anti-5-methylcytosine antibody. The ratio or amount of methylated DNA, which is proportional to the enzyme activity, can then be fluorometrically measured by reading the fluorescence intesntiy in a fluorescence microplate spectrophotometer at excitation 530 +/-20 nm and emission 580 +/-20 nm. The activity of DNMT enzymes is proportional to the RFU (relative fluorescence units) measured.
Easy, Fast, & FlexibleThe entire fluorometric assay has easy-to-follow steps for convenience and speed, which can be completed in 3 hours and 45 minutes. The stripwell microplate format allows for a flexible assay in manual or high throughput analysis.
Safe & ConvenientAll the needed reagents, including negative controls and positive controls, for measurement of DNMT activity/inhibition are conveniently packaged in the kit. The direct fluorometric quantification of DNA samples replaces obsolete or inferior methods and eliminates the need for radioactivity, extraction, or chromatography.
Highly Sensitive & SpecificThe novel procedure and proprietary kit compositions allow for an accurate measurement of DNMT activity/inhibition to be achieved with high sensitivity and specificity. The detection limit of the input DNA can be as low as 0.2 µg of nuclear extract or 0.2 ng of purified enzymes, which is ten times better than the predecessor kit.
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