The EpiQuik™ DNMT (DNA Methyltransferase) Activity/Inhibition Assay Ultra Kit is a complete set of optimized buffers and reagents that allows the experimenter to colorimetrically 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 five times more sensitive. The kit has the following advantages and features:
- Colorimetric assay with easy-to-follow steps for convenience and speed. The entire procedure can be completed within 3 hours and 45 minutes.
- Safe and innovative colorimetric assay without radioactivity, extraction, and chromatography.
- The ultra-sensitive detection limit can be as low as 0.5 µg of nuclear extract or 0.5 ng of purified enzymes, which is five times better than the predecessor kit.
- Optimized antibody & enhancer solutions allow high specificity to 5-mC without cross-reactivity to unmethylated cytosine.
- Strip-well microplate format allows for either low or high throughput analysis.
The EpiQuik Nuclear Extraction Kit has also been optimized for use with this kit.
About DNA Methyltransferases
The 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 colorimetric assay: (a) High sensitivity and specificity achieved by using recombinant DNMT1 with the EpiQuik DNMT Activity/Inhibition Assay Ultra Kit (Colorimetric); (b) High sensitivity and specificity achieved by using nuclear extract with the EpiQuik DNMT Activity/Inhibition Assay Ultra Kit (Colorimetric). Nuclear extracts were prepared from MCF-7 cells using the EpiQuik Nuclear Extraction Kit (Cat. No. OP-0002).
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 colorimetrically measured through an ELISA-like reaction by reading the absorbance in a microplate spectrophotometer at a wavelength of 450 nm. The activity of DNMT enzymes is proportional to the intensity of optical density measured.
Easy, Fast, & Flexible
The entire colorimetric 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 colorimetric 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 0.5 µg of nuclear extract or 0.5 ng of purified enzymes, which is five times better than the predecessor kit.
MU1 (10X Wash Buffer)
MU2 (DNMT Assay Buffer)
MU3 (Adomet, 50X)*
MU4 (DNMT Enzyme Control, 50 µg/ml)*
MU5 (Capture Antibody, 1000 µg/ml)*
MU6 (Detection Antibody, 400 µg/ml)*
MU7 (Enhancer Solution)*
MU8 (Developer Solution)
MU9 (Stop Solution)
8-Well Assay Strips (With Frame)
Adhesive Covering Film
*For maximum recovery of the products, centrifuge the original vial after thawing prior to opening the cap.