Overview

  • Product name
    Hydroxymethylated DNA Immunoprecipitation (hMeDIP) Kit
    See all hydroxymethylated DNA kits
  • Assay time
    3h 00m
  • Species reactivity
    Reacts with: Mouse, Human
  • 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 Immunoprecipitation (hMeDIP) Kit (ab117134) enables the user to enrich hydroxymethated DNA using an antibody specific to hydroxymethylcytosine (5-hmC) to immunoprecipitate hydroxymethylated genomic DNA hMeDIP. The enriched hydroxymethylated fractions can be used in various downstream applications including PCR (hMeDIP-PCR) and microarrays (hMeDIP-chip) for investigating gene-specific hydroxymethylation in cells or tissues.

Properties

Images

  • Sensitive detection of gene-specific hydroxymethylation by hMeDIP-QPCR.

    Human brain DNA (500 ng) was fragmented to 200-600 bps with a sonicator. The fragmented DNA was used for hydroxymethylated DNA enrichment with ab117134. Eluted DNA was analyzed by real time PCR with primers specifically for OCT4 or GAPDH sequences in the promoter regions. Results show that the promoter region is hydroxymethylated in OCT4 but not in GAPDH.
  • Selective enrichment of hydroxymethyated DNA with ab117134.

    50 pg of unmethylated, methylated, and hydroxymethylated DNA control were spiked into fragmented human genomic DNA (500 ng). hMeDIP was processed using 5-hmC antibody and non-immune IgG included in the kit. Eluted DNA was analyzed by real time PCR using control primers included in the kit to detect the presence of spiked control DNA.

Protocols

References

This product has been referenced in:
  • Yilmaz-Ozcan S  et al. Epigenetic mechanisms underlying the dynamic expression of cancer-testis genes, PAGE2, -2B and SPANX-B, during mesenchymal-to-epithelial transition. PLoS One 9:e107905 (2014). Functional Studies ; Human . Read more (PubMed: 25229454) »
See 1 Publication for this product

Customer reviews and Q&As

1-2 of 2 Abreviews or Q&A

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.

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Answer

Thank you for contacting us.

Ab117134 is shipped in blue ice at 4°C. Dry ice is not required for this particular kit. However, upon arrival, component EMH7 should be stored at 20°C away from light according to the datasheet instructions.

I hope this helps. Please do not hesitate to contact us for further assistance.

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