Overview

  • Product name
    Hydroxymethylated DNA Quantification Kit (Fluorometric)
    See all hydroxymethylated DNA kits
  • Detection method
    Fluorescent
  • Sample type
    Saliva, Urine, Plasma, Tissue, Adherent cells, Suspension cells
  • Assay type
    Quantitative
  • Sensitivity
    >= 10 pg/well
  • Assay time
    3h 20m
  • Species reactivity
    Reacts with: Other species, Mammals
  • Product overview

    DNA methylation is a covalent modification of the cytosine ring at the 5' position, 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. The biological important of 5-mC as a major epigenetic modification in phenotype and gene expression has been recognized widely. Quite recently, a novel modified nucleotide, 5-hydroxymethylcytosine (5-hmC), has been detected to be abundant in mouse brain and embryonic stem cells. It is a hydroxylated and methylated form of cytosine. Although it is still uncertain its specific role in epigenetics, it is believed it plays a role in DNA demethylation, chromatin remodeling and gene expression regulation.

    Hydroxymethylated DNA Quantification Kit (Fluorometric) (ab117131) is a complete set of optimized buffers and reagents to quantify global DNA hydroxymethylation by specifically measuring levels of 5-hydroxymethylcytosine (5-hmC) without cross-reactivity to methylcytosine and unmethylated cytosine.

    It can be paired to use with EpiSeeker methylated DNA Quantification Kit (Fluorometric) (ab117129), which specifically quantifies 5-mC, for simultaneously quantification of both methylated and hydroxymethylated DNA.

  • Platform
    Microplate reader

Properties

  • Storage instructions
    Please refer to protocols.
  • Components 48 tests 96 tests
    10X Wash Buffer 1 x 14ml 1 x 28ml
    8-Well Assay Strips (with Frame) 6 units 12 units
    Binding Solution 1 x 5ml 1 x 10ml
    Capture Antibody, 1000 µg/mL 1 x 4µl 1 x 8µl
    Detection Antibody, 400 µg/mL 1 x 8µl 1 x 16µl
    Enhancer Solution 1 x 8µl 1 x 16µl
    Fluoro Developer 1 x 8µl 1 x 16µl
    Fluoro Dilutor 1 x 4ml 1 x 8ml
    Fluoro Enhancer 1 x 8µl 1 x 16µl
    Negative Control I, 20 µg/mL 1 x 10µl 1 x 20µl
    Negative Control II, 20 µg/mL 1 x 10µl 1 x 20µl
    Positive Control, 20 µg/mL 1 x 10µl 1 x 20µl
  • Research areas

Images

  • Demonstration of high sensitivity and specificity of 5-hydroxymethylcytosine detection achieved using ab117131. Synthetic unmethylated DNA (contains only cytosine), methylated DNA (contains only 5-methylcytosine), and hydroxymethylated DNA standard (contains only 5-hydroxymethylcytosine) were added into the assay wells at different concentrations and then measured with ab117131.

Protocols

References

This product has been referenced in:
  • Pinzón-Cortés JA  et al. Effect of diabetes status and hyperglycemia on global DNA methylation and hydroxymethylation. Endocr Connect 6:708-725 (2017). Read more (PubMed: 28993426) »
See 1 Publication for this product

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

High variability

Average Good 4/5 (Ease of Use)
Abreviews
The kit is relatively easy to use and mercifully has less steps than the methylation assay kit ab117129.
There was high variability between my duplicate samples even though I followed the instructions very closely.

Abcam user community

Verified customer

Submitted Aug 20 2018

Answer

Both of these kits (ab117133 and ab117135) will immunoprecipitate methylated DNA. The ab117133 Kit uses purified DNA as starting materials while the ab117135 Kit uses cells as starting materials. I can confirm that the immunoprecipitated products are suitable to use for RT-PCR.

The 24 or 48 tests correspond to single immunoprecipitations and not to the number of samples that you are able to assay. Therefore, if for one cell type you need to do 6 tests, then you will be able to run 4 cell types with a 24 sample kit. The kits are in strip format though, so you can save what you don't use for a later time.

With regards to a hydroxymethylated DNA kit, we have https://www.abcam.com/episeeker-hydroxymethylated-dna-quantification-kit-colorimetric-ab117130.html, Seeker hydroxymethylated DNA Quantification Kit (Fluorometric) (ab117131) or https://www.abcam.com/episeeker-hydroxymethylated-dna-immunoprecipitation-hmedip-kit-ab117134.html.

The more suitable kit would depend on what type of information you want from your samples. The ab117130 and ab117131 kits will allow you to look at global hydroxymethylation levels of their samples and would tell you the percent of hydroxymethylation of your DNA but would not allow for RT-PCR. The kit ab117134 will allow the you to explore regions of hydroxymethylated DNA by immunoprecipitating/enriching the hydroxymethylated DNA followed by RT-PCR or hMeDIP-chip which is maybe more useful to you.

You can indeed purchase the anti-5-hydroxymethyl cytidine antibody (ab106918) and use it with the 5-mC immunoprecipitation kit since you coat the antibody on the wells.

Read More

Answer

Thank you for contacting Abcam regarding our EpiSeeker Hydroxymethylated DNA Quantification kits.


The main difference between the colorimetric and fluorometric kits is the method of detection - this will require either a microplate reader at 450nm or a fluorescence microplate reader at Ex/Em = 530/590 nm.


The sensitivies of these two kits is also quite different:


Colorimetric - 40pg of hydroxymethylated DNA from 50ng of input genomic DNA
Fluorometric - 10pg of hydroxymethylated DNA from 20ng of input genomic DNA


I hope this information is helpful. Please do not hesitate to contact me if you have any additional questions.

Read More

Answer


I would like to inform you that we are still investigating the problem you experienced with the kits. The lab has already provided the following information: "The reading can be carried out at 530/590 nm +/- 10 generally. Thus, emission at 595 nm is fine, and excitation at 550 may be Ok but suboptimal". From this Claudiarequested a graph of the excitiation and emission of the fluorochrome used in the kits and we are still waiting for more details. I have sent a reminder to the lab, we are very sorry we cannot still answer this question and I hope we will be able to do so within the next couple of days.

I appreciate the time you have spent on these experiments and would be pleased to arrange a replacement, a refund or credit note in compensation.As a replacement, please note thatthe only lots (for both kits)we currently have in stock are the same as the ones your received, let me know if you would like to receivefree of charge replacements and I will ask the source of the kits ifalternative lotsare available.

I look forward to hearing from you with details of how you would like to proceed.

Read More

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