Overview

  • Product name
    DNMT Activity Assay Kit (Colorimetric)
    See all DNMT kits
  • Detection method
    Colorimetric
  • Assay type
    Enzyme activity (quantitative)
  • Assay time
    3h 45m
  • Species reactivity
    Reacts with: Mouse, Rat, Human, Drosophila melanogaster, Plants
    Predicted to work with: Mammals, Fungi
  • Product overview

    DNMT Activity Quantification Kit (Colorimetric) ab113467 allows the user to colorimetrically measure DNA methyltransferase activity or inhibition at extremely fast speed on a 96-strip well 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.


    In the DNMT assay protocol, DNMT enzymes in a sample transfer methyl groups from Adomet to a DNMT DNA substrate coated onto microplate wells. The methylated DNA is bound with a 5-methylcytosine antibody. A secondary antibody binds the 5-methylcytosine antibody, and the enzyme-conjugated secondary antibody is detected using a chromogenic enzyme substrate.


    DNMT assay protocol summary:
    - add samples, positive controls, and samples with inhibitor to wells
    - incubate for 90-120 min, and wash wells three times with wash buffer
    - add capture antibody, incubate for 60 min, and wash wells
    - add detection antibody, incubate for 30 min, and wash wells
    - add enhancer solution, incubate for 30 min, and wash wells
    - add developer solution, incubate for 1-10 min
    - add stop solution and analyze with a microplate reader

  • Notes

    DNMT (DNA methylases) is a family of enzymes responsible for the addition of methyl groups on the cytosine ring at the 5' position of a CpG dinucleotide. The DNMT family is required for the establishment and maintenance of DNA methylation patterns.

  • Platform
    Microplate reader

Properties

Images

  • Demonstration of high sensitivity and specificity of the DNMT activity/inhibition assay achieved by using recombinant DNMT1 with ab113467.
  • Demonstration of high sensitivity and specificity of the DNMT activity assay achieved by using nuclear extracts with ab113467. Nuclear extracts were prepared from MCF-7 cells using ab113474.

Protocols

References

This product has been referenced in:
  • O'Leary VB  et al. Long non-coding RNA PARTICLE bridges histone and DNA methylation. Sci Rep 7:1790 (2017). Functional Studies ; Human . Read more (PubMed: 28496150) »
  • Panikar CS  et al. Presence of DNA methyltransferase activity and CpC methylation in Drosophila melanogaster. Mol Biol Rep N/A:N/A (2015). Functional Studies ; Drosophila melanogaster . Read more (PubMed: 26547851) »
See all 5 Publications for this product

Customer reviews and Q&As

Filter by Ratings

1-2 of 2 Abreviews

DNMT ACTIVITY CUANTIFICATION KIT
PROTOCOL for ADIPOSE TISSUE PROTEINS
• EDM1, EDM2, EDM3 were prepared following manual instructions.
• The amount of protein obtained in the nuclear extraction of this kind of tissue was very low. Therefore, we decided to use 20 g of nuclear extract and complete with a total volume of 100 L/well. (After different probes we saw that we need this amount of protein, and we safe the proportion, 1-5 L of nuclear extract and 40-45 L of EDM3, completing with 100 L/well.)
• For blank and positive control wells, the volume was changed to 100 L/well.
• The strip-well microplate was covered with the adhesive film and was incubated in an oven at 37ºC with continued shaking during 3 hours.
• The washes were done following the instructions in all steps.
• EDM5 were prepared in a dilution rate of 1:500 and incubated in the same conditions that in the previous step (oven at 37ºC with continued shaking during 1 hour).
• EDM6 were prepared in a dilution rate of 1:1000 and incubated in the same conditions that in the previous step (oven at 37ºC with continued shaking during 30minutes).
• EDM7 were prepared in a dilution rate of 1:2000 and incubated in the same conditions that in the previous step (oven at 37ºC with continued shaking during 30minutes).
• 100 L/well of EDM8 were added and the microplate was incubating at room temperature away from direct light about 30 minutes.
• EDM9 were added to stop the enzyme reaction.
• Activity calculation was done following the instructions.

Miss. Ana Jadraque

Verified customer

Submitted Jun 25 2014

DNMT activity in mouse brain

Good Good 4/5 (Ease of Use)
Abreviews
The kit is easy to use and contains all components needed to perform the colorimetric ELISA. Mouse brain samples were used with this kit. Nuclear extract was prepared (with a non abcam kit) from the previous collected mouse brain samples. From each sample 15 µg of nuclear extract was used to perform the ELISA. From here on the abcam protocol was followed. The blank an positive controls were included, and the positive control had the highest signal in my study design. Between different individual animals there was a low variation, speaking for the high sensivity and reproducibility of the kit.

Abcam user community

Verified customer

Submitted Jun 07 2013

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