The EpiQuik™ DNA Demethylase Activity/Inhibition Assay Ultra Kit is a complete set of essential components which enables an experimenter to measure total DNA demethylase activity/inhibition. The kit can be used with nuclear extracts from a broad range of species such as mammals and plants, in a variety of forms including, but not limited to, cultured cells and fresh tissues. The EpiQuik™ DNA Demethylase Activity/Inhibition Assay Ultra Kit has the following features:

• Colorimetric assay with easy-to-follow steps for convenience and speed. The entire procedure can be completed within 4 hours.
• Safe and innovative colorimetric assay without the need for radioactivity, extraction, or chromatography.
• An ultra-sensitive detection limit, as low as 1 µg of nuclear extract, which is five times more sensitive than the predecessor kit.
• 96 strip-well microplate format allows for either low or high throughput analysis.

Background Information
DNA demethylation is necessary for the epigenentic reprogramming of genes and is also directly involved in many important disease mechanisms such as tumor progression. Demethylation of DNA can either be passive or active, or a combination of both. Passive DNA demethylation usually takes place on newly synthesized DNA strands via DNMT1 during replication rounds. Active DNA demethylation mainly occurs by the removal of 5-methylcytosine through further modified cytosine bases which have been converted by TET enzyme-mediated oxidation. These oxidation products have been shown to be repaired by TDG, a glycosylase which is involved in base excision repair, or directly converted to cytosine by DNMT3A/ DNMT3B in oxidative states. It is proposed that DNA demethylation could also be initiated by deamination of 5-mC through candidate deaminases including AID and APOBEC1, which convert 5-mC to thymine. The resulting thymine could be repaired by BER initiated by a T-G mismatch glycosylase such as MBD4 or TDG. In addition, the 5-mC base can be directly removed in plants by the DME/ROS1 family of 5-mC DNA glycosylases, resulting in an abasic site that is repaired by the BER process.

Principle & Procedure
In an assay with this kit, the unique methylated DNA substrate is stably captured on the strip wells. Active DNA demethylases bind to and demethylate the DNA substrate. The methylated DNA can be recognized with a high affinity 5-methylcytosine antibody and the immuno-signal is enhanced with enhancer solution. The ratio or amount of methylated DNA, which is inversely proportional to enzyme activity, can then be colorimetrically quantified through an ELISA-like reaction. 

Starting Materials & Input Amount
The amount of nuclear extracts for each assay can be from 2 µg to 20 µg with an optimal range of 5 to 10 µg.  We recommend using Epigentek's nuclear extraction kit for optimal results.

Fig. 1. Schematic procedure of the EpiQuik™ DNA Demethylase Activity/Inhibition Assay Ultra Kit.

Fig. 2. Assay control standard was added into the assay wells at different concentrations and then measured with the DNA Demethylase Activity/Inhibition Assay Ultra Kit.