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MethylFlash Urine 5-Methylcytosine (5-mC) Quantification Kit (Colorimetric)


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Suggested Workflow
DNA Isolation
DNA Methylation Quantification
Schematic procedure for the MethylFlash™ Urine 5-Methylcytosine Quantification Kit (Colorimetric).
5-mC standard was added into the assay wells at different concentrations and then measured with the MethylFlash™ Urine 5-Methylcytosine Quantification Kit (Colorimetric).
5-mC level is quantified from different volumes of human urine using the MethylFlash™ Urine 5-Methylcytosine Quantification Kit (Colorimetric).
Input Type: Urine
Research Area: DNA Methylation
Target Application: Amount Quantitation
Vessel Format: 96-Well Plate
100% Guarantee: 6 months
Catalog No.SizePriceQty
P-1039-4848 assays $294.00 
P-1039-9696 assays $509.00 
Order now & get it by Friday, August 23rd  
Product Overview

The MethylFlash™ Urine 5-Methylcytosine (5-mC) Quantification Kit (Colorimetric)  is a complete set of optimized buffers and reagents to colorimetrically quantify 5-mC in urine using an inhibitory competitive immunoassay method. It is suitable for detecting total urinary 5-methylcytosine levels, resulting from whole body turnover or degradation of methylated DNA/RNA, in urine from humans and animals. The urine samples can be in fresh or frozen form. The kit has the following advantages and features:

  • Innovative colorimetric assay with easy-to-follow steps for convenience and speed. The entire procedure can be finished within 4 hours.
  • 96 strip-well microplate format makes the assay flexible: manual or high throughput analysis.
  • Innovative kit composition enables background signals to be extremely low, which eliminates the need for plate blocking and allows the assay to be simple, accurate, reliable, and consistent.
  • The level of 5-mC measured in human urine samples using this kit is comparable to that detected by HPLC method. 
  • A novel assay principle allows high sensitivity to be achieved. The detection limit can be as low as 0.03 ng/assay well or 1 nM of 5-mC.
  • Low input range of urine for each assay with a volume of 0.5 to 5 µl and an optimal volume of 1 µl.
  • Optimized antibody and enhancer solutions allow for high specificity to 5-mC, without cross-reactivity to unmethylated cytosine. 
  • Negative control and positive standard are included, which are suitable for quantification of 5-mC in free form and 5-mC contained in methylated DNA/RNA fragments from different urine samples.

Background Information
Nucleobase 5-methylcytosine (5-mC), a modified form of cytosine converted by cytosine methyltransferases, is widespread both in DNA and different cellular RNAs. The biological importance of DNA 5-mC methylation as a major epigenetic modification in phenotype and gene expression has been widely recognized. Recent data strongly suggests that RNA 5-mC methylation is also involved in the regulation of various biological processes including tRNA stability/recognition and mRNA translation. 

Urinary excretion of 5-mC including both 5-methyl-2-deoxycytidine and 5-methylcytidine is an indication of a whole body turnover or degradation of methylated DNA and RNA. The urinary 5-mC level can be altered by a change of the bodies’ turnover of methylated DNA/RNA or alteration of cellular DNA/RNA methylation status. A number of studies have indicated that 5-mC excreted in urine has the potential to act as a cancer biomarker, with an increased level arising from disease onset and progression. For example, an elevated level of urinary 5-mC was observed in lung cancer, breast cancer, and leukemia patients with active disease states. It was also shown that urinary 5-mC excretion is effected by Alzheimer’s disease or by radiation treatment. It is suggested that urinary 5-mC might be applicable as a biological marker for detecting some types of cancer and monitoring cancer progression after treatment with radiation or demethylation reagents. 

Chromatography-based techniques such as HPLC and TLC mass spectrometry are commonly used for detecting 5-mC in urine and although fairly acurate, are time consuming, less sensitive, and have low throughput with high costs. To address this problem, Epigentek offers the MethylFlash Urine 5-Methylcytosine (5-mC) Quantification Kit (Colorimetric) to quantify 5-mC or body turnover status of methylated DNA/RNA using urine samples.

Principle & Procedure
In this ELISA-like inhibitory competitive immunoassay assay, urine samples and the 5-mC standard are first incubated with a 5-mC antibody solution and then transferred to strip wells coated with methylated DNA (5-mC). The well is washed to remove any unbound reagents after incubation and then a detection antibody is added to generate a signal that can be measured colorimetrically by reading the absorbance in a microplate spectrophotometer. Because 5-mC in the urine sample inhibits the binding of 5-mC antibody to 5-mC coated on the well, higher concentrations of 5-mC in the urine sample lead to a reduced binding of the antibody to the 5-mC on the well. Therefore the signal or OD intensity measured from the well will be inversely proportional to the amount of 5-mC in the urine sample and the amount of 5-mC in the urine sample can be quantified by a comparison with a predetermined 5-mC standard.

Product Components

WB (10X Wash Buffer)
BS (Binding Solution)
MS (5-mC Standard, 4 µg/ml)*
ME3 (Negative Control, 50 µg/ml)*
ME4 (5-mC DNA solution, 320X)*
ME5 (Capture Antibody, 1000X)*
ME6 (Detection Antibody, 2000X)*
ES (Enhancer Solution)*
DS (Developer Solution)
SS (Stop Solution)
Plate 1 (Assay Plate) (With Frame)
Plate 2 (Sample Preparation Plate)
User Guide

* Spin the solution down to the bottom prior to use.


User Guide & MSDS

[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 along with your contact information and institution name.

[Safety Data Sheet]
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