The EpiQuik™ DNA Methyltransferase Activity/Inhibition Assay Kit is a convenient package of tools that allows the experimenter to measure Dnmt activity or inhibition at tremendously fast speeds and consistency; superior and safer than all other current methods. 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™ DNA Methyltransferase Activity/Inhibition (DNMT) Assay Kit is suitable for measuring Dnmt activity/inhibition from a broad range of species including mammalian cells/tissues, plants, and bacteria.

WHY CHOOSE THE EPIQUIK™ DNA METHYLTRANSFERASE ACTIVITY/INHIBITION ASSAY KIT?

• Very rapid procedure, which can be completed within 3 hours.
• Safe and innovative colorimetric assay without radioactivity, extraction, and chromatography.
• Strip microplate format makes the assay flexible: manual or high throughput analysis.
• Extremely simple, reliable, and consistent assay conditions.

The EpiQuik™ DNA Methyltransferase Activity/Inhibition Assay Kit is designed for measuring total Dnmt activity (de novo, maintenance). In an assay with this kit, the unique cytosine-rich DNA substrate is stably coated on the strip wells. These wells are specifically treated to have a high DNA absorption ability. Dnmt enzymes transfer a methyl group to cytosine from Adomet to methylate 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 enzyme activity, can then be colorimetrically quantified through an ELISA-like reaction. SCHEMATIC PROCEDURE:

Analysis of Dnmt activity in Hela nuclear extract. HSC-3 cells were incubated with/without zebularine (220 mM) for 48 hours. Nuclear proteins were extracted and total Dnmt activity was measured.

M1 (10X wash buffer)
M2 (Dnmt assay buffer)
M3 (Adomet, 8 mM)*
M4 (Dnmt positive control)*
M5 (capture antibody)*
M6 (detection antibody 200 µg/ml)*
M7 (developing solution)
M8 (stop solution)
8 Well substrate-coated strip (with frame)
User guide

* For maximum recovery of the products, centrifuge the original vial after thawing prior to opening the cap.

1. Can protein extracts from frozen tissue stored at -80°C be used with this kit?
No, protein extracts should be used from fresh tissue.

2. What is the minimum amount of Dnmt protein required for a successful assay?
The minimum amount of Dnmt protein is about 0.2 µg for a successful assay.

3. Do I have to do all the assays at once with the kit or can I do them in portions?
Yes, you can do them in portions by using only the reagent amounts you need, including diluting M5 and M6 as needed.

4. How do you aspirate the washing solution from the wells?
By using pipette tips.

5. How long can M5 and M6 be stored for after dilution?
1 or 2 days.

6. Can this kit distinguish Dnmt1 and Dnmt3a/3b?
The P-3001 kit cannot distinguish between Dnmt1 and Dnmt3a/3b. It will work well with both enzymes.

7. How are the wells coated?
The wells are coated with a proprietary Dnmt binding substance (high affinity).

8. Is there any reason why I cannot start with recombinant DNMT1, incubate with my inhibitors, and then use this to proceed with the assay?
This kit is suitable for measuring Dnmt activity of both cell extract and recombinant Dnmts (including Dnmt1) in the absence or presence of inhibitors. The capture mAb included in this kit is against converted (methylated) substrate.

[User Guide]* *Always use the actual User Guide that shipped with your product. Is the above file locked? You can also request user guides by emailing info@epigentek.com along with your contact information and institution name.

[Material Safety Data Sheet]

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