DNMT Activity / Inhibition Assay

Synonyms

DNA methyltransferase, methyltransferase, MTase, DNMT, DNMT1, DNMT3a, DNMT3b, activity assay, inhibition assay, non-radioactive, MBD, methyl CpG binding protein, CpG, methylation, methyl, DNA methylation, de novo methyltransferase, maintenance methyltransferase

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Overview

The DNMT Activity / Inhibition Assay is a non-radioactive assay to measure the activity or inhibition of DNA methyltransferases. The sensitive ELISA-based method utililzes the high affinity binding of methyl CpG binding domain (MBD) protein towards methylated DNA in order to detect DNA methyltransferase activity on the provided CpG-enriched DNA substrate. The assay is performed in a 96-stripwell plate, enabling low or high throughput screening that is easily quantified by spectrophotometry. For complete details, click the Assay Method tab below.

The DNMT Activity / Inhibition Assay provides everything needed to study DNA methyltransferase activity from recombinant DNMT enzymes (DNMT1, DNMT3a & DNMT3b) or nuclear extract samples. A CpG Methyltransferase is included as a positive control. For added convenience, nuclear extracts can be prepared using Active Motif's Nuclear Extract Kit. Click the DNMT Info tab for data and more information; kit manuals can be downloaded under the Documents tab.

DNMT Info

DNMT Activity / Inhibition Data

Figure 1: DNMT activity using a CpG Methyltransferase.

The DNMT Activity / Inhibition Assay was used to generate a standard curve from 0.125 - 2 units per well using the included CpG Methyltransferase control. The assay was able to detect DNMT activity from as little as 0.125 units of enzyme with a 1.5 hour incubation time and a 5 minute developing time. Data shown are the results from wells assayed in duplicate. These results are provided for demonstration only.

Figure 2: DNMT Activity from MCF-7 nuclear extracts.

The DNMT Activity / Inhibition Assay was used to screen MCF-7 nuclear extracts (0.625 - 10 µg) prepared using Active Motif's Nuclear Extract Kit (Catalog No. 40010). The assay was able to detect DNMT activity from as little as 0.625 µg of DNMT with a 1.5 hour incubation time and a 5 minute developing time. Data shown are the results from wells assayed in duplicate. These results are provided for demonstration only.

Figure 3: DNMT Inhibition of MCF-7 cells with 5-Aza 2´ deoxycytidine.

The DNMT Activity / Inhibition Assay was used to screen for DNMT inhibition in MCF-7 cells that were either untreated or treated with 5-Aza 2´ deoxycytidine for 3 - 5 days. Nuclear extracts were prepared using Active Motif's Nuclear Extract Kit (Catalog No. 40010) and 5 µg of each treatment condition were tested in the assay with a 1.5 hour incubation time and a 3 minute developing time. The 5-Aza 2´ deoxycytidine treatment showed a 25% inhibition of DNMT activity as compared to the mock treated sample for the 5 day treatment. Data shown are the results from wells assayed in duplicate. These results are provided for demonstration only.

DNA Methyltransferase (DNMT) Info

DNA methylation is a major epigenetic modification in the genome of higher eukaryotes. This DNA covalent modification is catalyzed by DNA methyltransferase enzymes (DNMTs or DNA MTases) and consist in the addition of a methyl group from S-adenosyl-L-methionine (AdoMet) to the fifth carbon position of cytosine (cytosine-5 or C5), mostly within CpG dinucleotides. This methylation, together with histone modifications, plays an important role in modulating chromatin structure, thus controlling gene expression and many other chromatin-dependent processes.

DNA methylation is accomplished by three enzymes: DNMT1, DNMT3a and DNMT3b. These enzymes can be further classified as de novo methyltransferases, enzymes that are able to methylate previously unmethylated CpG sequences, or maintenance methyltransferases, which copy pre-existing methylation marks onto new DNA strands during replication^1,2. DNMT1 is the most abundant DNA methyltransferase in mammalian cells and predominatly methylates hemimethylated CpG dinucleotides as a maintenance methyltransferase, but DNMT1 has also been shown to function as a de novo DNA methyltransferase^3,4,5. DNMT3a methylated CpG dinucleotides at a slower rate than DNMT1, but a greater rate than DNMT3b.

In addition to DNMT1, DNMT3a and DNMT3b there are two non-canonical family members, DNMT2 and DNMT3L. DNMT2 is not a DNA methyltransferase, it methylates cytosine 38 in the anticodon loop of tRNA16 but does not methylate DNA. DNMT3L is closely related to DNMT3a and DNMT3b structurally, but is catalytically inactive as a DNA methyltransferase. DNMT3L is known to associate with both DNMT3a and DNMT3b and may be responsible for the recruitment of histone deacetylases to direct repression onto newly established imprints⁶.

References

1. Okano M. et al. (1999) Cell, 99:  247-257
2. Jeltsch, A. (2006) Curr. Top. Microbiol. Immunol., 301:  203-225
3. Fatemi et al. (2002) Eur. J. Biochem., 269:  4981-4984
4. Gowher, H. et al. (2005) Biochemistry, 44:  9899-9904
5. Liang et al. (2002) Mol. Cell Biol., 22:  480-491
6. Deplus, R. et al. (2002) Nucleic Acids Res., 30:  3831-3838

Assay Methods

DNMT Activity / Inhibition Activity Method

The DNMT Activity / Inhibition Assay is a time-saving, non-radioactive assay to measure DNA methyltransferase activity and/or inhibition from recombinant DNMT enzymes (DNMT1, DNMT3a & DNMT3b) or nuclear extract samples. This sensitive ELISA-based method uses the ability of methyl CpG binding domain (MBD) proteins to bind methylated DNA with high affinity. In the DNMT assay method, a universal CpG-enriched DNA substrate has been immobilized on a 96-stripwell plate. Purified DNMTs or DNMT activities from nuclear extracts will catalyze the transfer of methyl groups from the provided AdoMet reagent to the coated DNA substrate. The resulting methylated DNA will be recognized by the His-tagged recombinant MBD2b in an amount proportional to the enzyme activity. Addition of a polyHistidine antibody conjugated to horseradish peroxidase (HRP) provides a sensitive colorimetric readout that is easiy quantified by spectrophotometry. The assay includes a CpG Methyltransferase enzyme as a positive control.

DNA Methyltransferase Activity Assay Advantages

• Non-radioactive – colorimetric assay is easily quantified by spectrophotometry on a microplate reader at 450 nm
• Sensitive – unique methyl CpG binding domain (MBD) protein approach enhances the sensitivity of detection from either purified DNMT proteins or nuclear extracts
• Fast – assay can be completed in less than 3 hours
• Less effort required – kit is compatible with multi-channel pipettors to streamline wash steps
• Flexible – stripwell plate allows screening in low or high throughput

Contents

Contents & Storage

DNMT Activity / Inhibition Assay includes a 96-stripwell Assay plate, anti-polyHis-HRP antibody, Enzymatic Buffer AM1, 100X AdoMet, His-MBD2b protein, Binding Buffer AM11, 10X Wash Buffer AM1, 10X Wash Buffer AM3, 10X Antibody Binding Buffer AM3, Developing Solution, Stop Solution, CpG Methyltransferase (control) and plate sealer. Storage conditions vary from room temperature to -20°C, see manual for details. All reagents are guaranteed stable for 6 months when stored properly.

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