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

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

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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 $318.00 
P-1032-9696 reactions $559.00 
Availability: Usually Ships in 1 Day or Same day delivery Same Day NY Delivery 
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.

  • 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

Background
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. 

Product Components

WB (10X Wash Buffer)
BS (Binding Solution)
NC (Negative Control with 0% 5-hmC, 50 µg/ml)*
PC (Positive Control contains 1% 5-hmC, 50 µg/ml)*
hmAb (5-hmC 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 unhydroxymethylated DNA containing 0% of 5-hydroxymethylcytosine. The PC is hydroxymethylated DNA containing 1% of 5-hydroxymethylcytosine.

 

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 m*******@oregonstate.edu 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
Pros:
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.

Cons:
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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