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For high-throughput, detection of global 5-methylcytosine (5-mC) in DNA.
The streamlined workflow can be completed in less than 3 hours.

Product Size Catalog # Price Qty
5-mC DNA ELISA Kit 1 x 96 D5325
5-mC DNA ELISA Kit 2 x 96 D5326

About 5-mC DNA ELISA Kit

The ability to efficiently detect and quantify DNA methylation (i.e., 5-methylcytosine) has become essential for epigenetic-based research.  To date, a number of methods have been developed for this purpose including high-performance capillary electrophoresis, bisulfite sequencing, and methylated DNA immunoprecipitation.

The 5-mC DNA ELISA Kit is a convenient and powerful tool that allows the researcher to accurately quantitate 5-mC in any DNA sample in less than 3 hours. The kit features a unique Anti-5-Methylcytosine monoclonal antibody that is both sensitive and specific for 5-mC.  The assay is compatible with a wide range of input DNA from vertebrate, plant, and microbial sources as well as PCR amplicons and fragmented DNA. Percent 5-mC in a DNA sample can be accurately quantified from a standard curve generated with specially designed controls included with the kit.  Also, the fast, streamlined workflow is ideal for high-throughput analyses.

Equipment Incubator and ELISA plate reader. A multi-channel pipettor is recommended. An automated plate washer may be used for blocking and wash steps.
DNA Size Limits This protocol is optimized for 100 ng input DNA/well. Compatible with DNA in the range of 10-200 ng.
Sample Sources Purified genomic DNA, plasmid DNA, PCR amplification products, or DNA fragments in water, Tris-EDTA, or similar.
Detection ≥ 0.5% 5-methylcytosine (5-mC) per 100 ng single-stranded DNA.

This protocol is optimized for 100 ng of DNA per well.

Duplicate samples are recommended for accurate 5-mC detection and quantification.

DNA Coating:

 1.  Remove the necessary number of well strips to assay DNA samples and controls.

2.  Add 100 ng of each DNA to a PCR tube and bring the final volume to 100 µl with 5-mC Coating Buffer.

Example:  If the DNA concentration is 20 ng/μl, add 5 μl of DNA to 95 μl of 5-mC Coating Buffer for a final volume of 100 μl.

3.  Denature the DNA at 98°C for 5 minutes in a thermal cycler.   After denaturation, transfer immediately to ice for 10 minutes.

4.  Add the denatured DNAs to the wells of the plate, cover with foil, and incubate at 37 ºC for 1 hour.


 1.  Discard the buffer from the wells.

2.  Wash each well 3 times with 200 μl of 5-mC ELISA Buffer.  Discard the buffer after each wash.

 3.  Add 200 μl of 5-mC ELISA Buffer to each well.  Cover the plate with foil and incubate at 37 ºC for 30 minutes.

Antibody Addition:

 1.  Discard buffer from the wells.

2.  Prepare an antibody mix5 consisting of Anti-5-Methylcytosine and Secondary Antibody in 5-mC ELISA Buffer according to the following table:



Volume (µl)

Example (18 wells)

5-mC ELISA Buffer


(# wells + 2) 100

2,000 µl



Buffer Vol. / 2,000

1 µl

Secondary Antibody


Buffer Vol. / 1,000

2 µl

3.  Add 100 μl of this antibody mix to each well. Cover the plate with foil and incubate at 37ºC for 1 hour.

Color Development:

1.  Discard the antibody mix from the wells.

2.  Wash each well 3 times with 200 μl of 5-mC ELISA Buffer.

3.  Add 100 μl of HRP Developer to each well.  Allow color to develop for 10-60 minutes6 at room temperature.

4.  Measure absorbance at 405-450 nm using an ELISA plate reader.

For specific notes and additional information, please see the product protocol PDF.
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Featured Citations

DNA methylation in mouse brain tissue was quantified using the 5-mC DNA ELISA kit after exposure to different diets. Authors found that if cystathionine-β-synthase (a deficiency in this enzyme can play a role hyperhomocysteinemia) was included in the diet , 5-mC levels increased. Folic acid supplemented in the diet did not change 5-mC levels.
The authors showed that exposure to house-dust mites (HDM) decreased global percentage of 5-mC but increased the percentage of 5-hmC detected in the lungs of mice compared to saline treated control group.
The 5-mC DNA ELISA Kit was used to determine the effect of treament of fetal cardiomyocytes with endothelin-1 (ET-1), a protein expressed during hypoxia on methylation levels. They found that ET-1 increased %5-mC, but 5-Aza-2’-deoxycytidine blocked ET-1 effects.
The authors used the 5-mC DNA ELISA kit to quantify global methylation status of DNA isolated for CD4+ T cells in Autoimmune Addison’s Disease (AAD) patients and healthy patients. AAD patients had significantly lower levels of 5-mC.
Authors used the 5-mC DNA ELISA kit to compare the levels of 5-mC in cardiomyocytes during hyperhomocysteinemia (HHcy), a condition that can lead to aberrant DNA methylation and histone acetylation and is associated with higher risk of neurovascular diseases. They found that 5-mC was increased in cardiomyocytes during HHcy compared to wild type cells.
The DNA methylation and hydroxymethylation levels in blood were determined in a number of blood samples taken from healthy men free of cardiovascular disease who participated in a study that sought to characterize metabolic abnormalities and subclinical atherosclerosis.
The 5-mC DNA ELISA Kit was used to quantify 5-mC levels in gliobastoma stem cells with and without treatment with temozolomide. They found that incubation of cells with temozolomide increased %5-mC by roughly 25%.

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