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MethylFlash Global DNA Methylation (5-mC) ELISA Easy Kit (Colorimetric)

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

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Suggested Workflow
DNA Isolation
 
 
DNA Methylation Quantification
 
Schematic procedure for the MethylFlash Global DNA Methylation (5-mC) ELISA Easy Kit (Colorimetric).
An example of an optimal standard curve generated with 5-mC standard control.
Accurate quantification of 5-mC content of various DNA samples from different species using the MethylFlash Global DNA Methylation (5-mC) ELISA Easy Kit (Colorimetric). The results are closely correlated with those obtained by HPLC-MS.
Input Type: DNA
Research Area: DNA Methylation
Target Application: Amount Quantitation
Vessel Format: 96-Well Plate
100% Guarantee: 6 months
Catalog No.SizePriceQty
P-1030-4848 reactions $299.00 
P-1030-9696 reactions $539.00 
Availability: Usually Ships in 1 Day or Same day delivery Same Day NY Delivery 
Product Overview

The MethylFlash™ Global DNA Methylation (5-mC) ELISA Easy Kit  is a complete set of optimized buffers and reagents to colorimetrically quantify global DNA methylation status by specifically measuring levels of 5-methylcytosine (5-mC) in a simplified, "one-step" ELISA-like reaction. As a fourth generation technology of Epigentek's patented global DNA methylation technique, it is a further refinement of the popular, 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 Hydroxymethylation (5-hmC) 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 0.05% methylated DNA from 100 ng of input DNA
  • Specific - High specificity to 5-mC, with no cross-reactivity to unmethylated cytosine and no cross-reactivity to hydroxymethylcytosine within the indicated concentration range of the sample DNA
  • Universal - Positive and negative controls are included and allow for detection of DNA methylation 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

Background
DNA methylation occurs by the covalent addition of a methyl group at the 5-carbon of the cytosine ring by DNA methyltransferases, resulting in 5-methylcytosine (5-mC). In somatic cells, 5-mC is found almost exclusively in the context of paired symmetrical methylation of the dinucleotide CpG, whereas in embryonic stem (ES) cells, a substantial amount of 5-mC is also observed in non-CpG contexts. Levels of 5-mC are variable in animal genomes, ranging from undetectable amounts in some insects to about 2% of total DNA in vertebrates. The level of 5-mC in plants generally accounts for 0.5-2% and can be as high as 8% of total DNA in some other species.  The biological importance of 5-mC as a major epigenetic modification in phenotype and gene expression has been recognized widely. For example, global decrease in 5-mC content (DNA hypomethylation) is likely caused by methyl-deficiency due to a variety of environmental influences, and has been proposed as a molecular marker in multiple biological processes such as cancer. It has been well demonstrated that the decrease in global DNA methylation is one of the most important characteristics of cancer. A few novel modified nucleotides, 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxycytosine (5-caC) have been detected in human and mouse tissues as well as embryonic stem cells. In mammals, these modified nucleotides can be generated by iterative oxidation of 5-methylcytosine, a reaction mediated by the TET family of enzymes.

Principle & Procedure
This kit contains all reagents necessary for the quantification of global DNA methylation. In this assay, DNA is bound to strip-wells that are specifically treated to have a high DNA affinity. The methylated fraction of DNA is detected using capture and detection antibodies and then quantified colorimetrically by reading the absorbance in a microplate spectrophotometer. The percentage of methylated 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. DNA can be isolated from any species such as mammals, plants, fungi, bacteria, and viruses in a variety of forms including, but not limited to, cultured cells, fresh and frozen tissues, paraffin-embedded tissues, plasma/serum samples, and body fluid samples. Both single stranded DNA and double stranded DNA with a size of 200 bps to full length is suitable for use.

Product Components

WB (10X Wash Buffer)
BS (Binding Solution)
NC (Negative Control with 0% 5-mC, 50 µg/ml)*
PC (Positive Control contains 5% 5-mC, 50 µg/ml)*
mcAb (5-mC Antibody, 1000X)*
SI (Signal Indicator, 1000X)*
ES (Enhancer Solution, 1000X)*
DS (Developer Solution)
SS (Stop Solution)
8-Well Assay Strips (With Frame)
User Guide

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

Note: The NC is unmethylated DNA containing 0% of 5-methylcytosine. The PC is methylated DNA containing 5% of 5-methylcytosine.

 

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 info@epigentek.com along with your contact information and institution name.

Customer Reviews

Rating by c*******@fiu.edu Verified Purchase Reviewed on: Wednesday 26 April, 2017
Application Description
Determination of seasonal changes in DNA methylation levels in gill tissue from the flat tree oyster Isognomon alatus sampled in the North Biscayne Bay, Florida.

Procedural Details
Flat tree oysters (I. alatus) were collected monthly over a 12-month span from ten distinct sites across North Biscayne Bay in North Miami, FL. Dissected gill tissue from five individual oysters per site and per month were pooled and genomic DNA extracted following an isopropanol-mediated method (Fernandez-Tajes et al. 2007).

Global Methylation was measured using 100 ng of isolated DNA following the manufacturer’s protocols.

Other Thoughts
Results across replicates were generally okay. Not too many outliers were detected.

We noticed that the kit reads methylation levels of intact and fragmented genomes as a few of our samples were slightly degradated but, still, the obtained results were consistent also in those samples.

The manual is also very detailed.

Overall, we are satisfied with the perfomance of this kit and plan to order it again anytime soon.
Rating by m*******@oregonstate.edu Verified Purchase Reviewed on: Tuesday 07 March, 2017
Application Description
Determining differences in global DNA methylation in zebrafish (Danio rerio) embryos spawned from adult fish maintained on a vitamin E sufficient (control) vs. vitamin E deficient diet.

Procedural Details
Data analyses with % 5m-C calculated according to P-1030 instructions; graphs and additional statistical analyses performed used GraphPad Prism 6.0 software.

Global DNA methylation in E-sufficient (E+) vs. E-deficient (E-) zebrafish embryos over 120 hours 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<0.01;**** p<0.0001 between groups at indicated age.

Other Thoughts
This was simple to use on the first try; product design and speed of protocol was much improved from the previous P-1034 model (used by another researcher at my institution).

How friendly was the user guide?
Most sections were easy to follow, and important protocol notes were highlighted or italicized for emphasis.

How professional was the appearance and presentation of the product?
The product was well organized and all components were clearly labeled.

How would you rate the overall product?
This product was well designed and easy to use, assuming one reviewed the protocol before beginning the experiment.

Pros:
Well-written protocol, straight-forward directions, and no "surprise" steps included out-of-order. All helpful details regarding each step were presented in a logical manner. There were sufficient reagent amounts to complete experiments with the total number of samples advertised.

Cons:
The actual plate with plastic wells was flimsy and it was difficult to extract the well rows without breaking them. Also, the instructions to mix standards (for quantification purposes) in 1.5 mL tubes could have been improved by instructing smaller tubes (i.e. microtubes) since the standard series volumes were so small. The micro-liter amounts got "lost" in 1.5 mL tubes.






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