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MethylFlash Global DNA Hydroxymethylation (5-hmC) ELISA Easy Kit (Colorimetric)


For quantitation of global DNA hydroxymethylation via a one-step ELISA method

Citations (14) | (1) | Write a Review
Suggested Workflow
DNA Isolation
DNA Methylation Quantification
Schematic procedure of the MethylFlash™ Global DNA Hydroxymethylation (5-hmC) ELISA Easy Kit (Colorimetric).
An example of an optimal standard curve generated with 5-hmC standard control.
Accurate quantification of 5-hmC content of various DNA samples from different species with the MethylFlash™ Global DNA Hydroxymethylation (5-hmC) ELISA Easy Kit (Colorimetric). The results are closely correlated with those obtained by MS-LC.
Input Type: DNA
Research Area: DNA Methylation
Target Application: Amount Quantitation
Vessel Format: 96-Well Plate
100% Guarantee: 6 months
Catalog No.SizePriceQty
P-1032-4848 reactions $329.00 
P-1032-9696 reactions $579.00 
Order within 10 hr 33 min & get it by tomorrow 
Product Overview

The MethylFlash™ Global DNA Hydroxymethylation (5-hmC) ELISA Easy Kit (Colorimetric) is a complete set of optimized buffers and reagents to colorimetrically quantify global DNA hydroxymethylation status by specifically measuring levels of 5-hydroxymethylcytosine (5-hmC) in a simplified, “one-step” ELISA-like format. As a fourth generation technology of Epigentek's popular global DNA methylation technique, it is a further refinement of the predecessor MethylFlash kit by improving upon speed, simplicity, sensitivity, and reproducibility.

This kit is also specifically optimized for paired use with the MethylFlash Global DNA Methylation (5-mC) ELISA Easy Kit (Colorimetric) for simultaneously quantifying both methylated DNA and hydroxymethylated DNA.

This kit has the following advantages and features:

  • Fast - Reduced steps so that the entire procedure only needs 2 hours*
  • Robust - Improved kit composition allows the assay to have a greater “signal window” with reduced variation between replicates
  • Convenient - Inherently low background noise, thereby eliminating the need for DNA denaturation and plate blocking steps
  • Sensitive - Detection limit can be as low as 0.01% hydroxymethylated DNA from 100 ng of input DNA
  • Specific - High specificity to 5-hmC, with no cross-reactivity to unmethylated cytosine or methylated cytosine within the indicated concentration range of the sample DNA
  • Universal – Positive and negative controls and allow detection of DNA hydroxymethylation in any species from either single-stranded or double-stranded input DNA
  • Accurate - Optimized positive controls that can be fractionalized in percentage scale, allowing the assay to be more accurate and highly comparable with HPLC-MS analysis
  • Flexible - Strip-well microplate format makes the assay available for manual or high throughput analysis

* Based on a single sample assay in duplicate

5-hydroxymethylcytosine (5-hmC), as a sixth DNA base with functions in transcription regulation, has been detected to be abundant in human and mouse brains and embryonic stem (ES) cells. In mammals, it can be generated by the oxidation of 5-mC, a reaction mediated by the ten-eleven translocation (TET) family of 5-mC-hydroxylases. 5-hmC has been demonstrated to be tissue specific, ranging from undetectable levels in cultured cell lines to 0.6% in human brain tissues, and can be as high as 8% of total DNA in some other species. The biological significance of 5-hmC as an important epigenetic modification in phenotype and gene expression has been recently recognized. For example, global decrease in 5-hmC content (DNA hypomethylation) has been exhibited in nearly all cancers and has been proposed as a molecular marker and therapeutic target in cancer as well. 

Principle & Procedure
This kit contains all reagents necessary for the quantification of global DNA hydroxymethylation. In this assay, DNA is bound to strip-wells that are specifically treated to have a high DNA affinity. The hydroxymethylated fraction of DNA is detected using a 5-hmC mAb-based detection complex in a one-step manner and then quantified colorimetrically by reading the absorbance in a microplate spectrophotometer. The percentage of hydroxymethylated DNA is proportional to the OD intensity measured.

Starting Materials
Input DNA should be relatively pure with 260/280 ratio >1.6 and can be diluted with water or TE buffer. The DNA amount can range from 20 ng to 200 ng per reaction. However, we recommend using 100 ng of DNA, which is the optimized input amount for the best results. 

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]
Product Citations

Sui C et. al. (November 2019). Electrochemiluminescence biosensor for DNA hydroxymethylation detection based on enzyme-catalytic covalent bonding reaction of -CH<sub>2</sub>OH and thiol functionalized Fe<sub>3</sub>O<sub>4</sub> magnetic beads. Biosens Bioelectron. :111908.

Guo W et. al. (November 2019). Homocysteine accelerates atherosclerosis by inhibiting scavenger receptor class B member1 via DNMT3b/SP1 pathway. J Mol Cell Cardiol.

Burghardt KJ et. al. (October 2019). Skeletal muscle DNA methylation modifications and psychopharmacologic treatment in bipolar disorder. Eur Neuropsychopharmacol.

Doherty TS et. al. (September 2019). DNA methylation and behavioral changes induced by neonatal spinal transection. Infant Behav Dev. 57:101381.

Sui C et. al. (July 2019). Photoelectrochemical biosensor for 5hmC detection based on the photocurrent inhibition effect of ZnO on MoS<sub>2</sub>/C<sub>3</sub>N<sub>4</sub> heterojunction. Biosens Bioelectron. 142:111516.

Cramer T et. al. (July 2019). Early-life epigenetic changes along the corticotropin-releasing hormone (CRH) gene influence resilience or vulnerability to heat stress later in life. Mol Psychiatry. 24(7):1013-1026.

Wu Y et. al. (May 2019). Genome-wide DNA methylation and hydroxymethylation analysis reveal human menstrual blood-derived stem cells inhibit hepatocellular carcinoma growth through oncogenic pathway suppression via regulating 5-hmC in enhancer elements. Stem Cell Res Ther. 10(1):151.

D'Aniello C et. al. (May 2019). Collagen prolyl hydroxylation-dependent metabolic perturbation governs epigenetic remodeling and mesenchymal transition in pluripotent and cancer cells. Cancer Res.

Németh CE et. al. (January 2019). Decreased Nuclear Ascorbate Accumulation Accompanied with Altered Genomic Methylation Pattern in Fibroblasts from Arterial Tortuosity Syndrome Patients. Oxid Med Cell Longev. 2019:8156592.

May JL et. al. (January 2019). IDH3α regulates one-carbon metabolism in glioblastoma. Sci Adv. 5(1):eaat0456.

Cramer T et. al. (December 2018). PARP Inhibitor Affects Long-term Heat-stress Response via Changes in DNA Methylation. Neuroscience. 399:65-76.

Jarome TJ et. al. (July 2018). EZH2 Methyltransferase Activity Controls Pten Expression and mTOR Signaling During Fear Memory Reconsolidation. J Neurosci.

McKee SE et. al. (February 2018). Perinatal high fat diet and early life methyl donor supplementation alter one carbon metabolism and DNA methylation in the brain. J Neurochem.

Lewinska A et. al. (February 2017). Downregulation of Methyltransferase Dnmt2 Results in Condition-dependent Telomere Shortening and Senescence or Apoptosis in Mouse Fibroblasts. J Cell Physiol.

Customer Reviews

Rating by m******* Verified Purchase Reviewed on: Wednesday 15 March, 2017
Application Description
The user guide was straight-forward and easy to interpret. Directions were clear and detailed. Some steps were ambiguous, however. For example, when preparing the diluted wash buffer solution in Step 1, the kit instructs to “adjust pH to 7.2-7.5” but does not give information as to how to do this, and whether doing so with concentrated acid or base solutions would affect the assay outcomes.

The product contents were well-packaged and secured in place; all materials were present in the specified amounts. Also, there was sufficient materials to perform the total number of experiments indicated by the kit (e.g. 48 or 96) – I did not run out of wash buffer, standards, etc. which is often the case with other kits from different companies.

Overall, this product was easy to use one the first trial, after having read the instruction guide and planning accordingly. Experiment outcomes were of acceptable quality and the kit worked as advertised. I was very happy with this product and would recommend it to other investigators.

Procedural Details
Determining differences in global DNA hydroxy-methylation in zebrafish (Danio rerio) embryos spawned from adult fish maintained on a vitamin E sufficient (control) vs. vitamin E deficient diet. Since vitamin E is an antioxidant, a greater amount of 5-hydroxymethylcytosine (5-hmC) expected in the E- group.

Global DNA methylation and hydroxy-methylation in E-sufficient (E+, blue) vs. E-deficient (E-, red-stripped) zebrafish embryos over 12 days post-fertilization (hpf); n= 15 embryos/sample; 4 replicate samples per group at each indicated age. p values from 2-way ANOVA with Tukey's post-test for multiple comparisons; * p<0.05, ** p<0.01;**** p<0.0001 between groups at indicated age.

Other Thoughts
Clear and concise instruction manual, detailed instructions, sample calculations, and sample/suggested plate arrangements for sample organization were all positive attributes of this kit. The user guide as very helpful and made performing the assay, and obtaining quality data, relatively easy to do.

Some instructions were ambiguous (see above regarding pH adjustment) and it would have been helpful to include certain details in different (earlier) steps – for instance, a reminder to turn on an incubator to 37C prior to beginning the assay, so that such would be ready by the time the experimenter needed to incubate the plate.

Also, the plastic wells and plate frame were flimsy and easily broke when trying to insert or remove the sample-well rows. The kit overall is of high quality, so making these items of higher-grade or thicker plastic would improve the product.

Calculated according to kit instructions; graphs and additional statistical analyses performed with GraphPad Prism 6.0 software.
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