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 & Procedure
In 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, & Flexible
The 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 & Convenient
All 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 & Specific
The 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.