5-hydroxymethylcytosine (5-hmC) Antibody (rabbit)

Catalog Number
50 μg
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Polyclonal antibody raised in rabbit against 5-hydroxymethylcytosine conjugated to KLH.

Concentration3.5 µg/µl
Species reactivityHuman, mouse, other (wide range)
PurityAffinity purified
PrecautionsThis product is for research use only. Not for use in diagnostic or therapeutic procedures.
Applications Suggested dilution References
hMeDIP* 3.5 μg/IP Fig 1
ELISA 1:100 - 1:500 Fig 2
Dot Blotting 1:1,1000 Fig 3
  • Validation Data


    Figure 1 hMeDIP results obtained with the Diagenode antibody directed against 5-hmC
    hMeDIP (hydroxymethylated DNA IP) was performed using the Diagenode antibody against 5-hydroxymethylcytosine (Cat. No. pAb-HMC-050). DNA from mouse ES cells was prepared with the GenDNA module of the hMeDIP kit and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. One μg of sheared DNA was spiked with the unmethylated (C) methylated (mC), and hydroxymethylated (hmC) controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack for hMeDIP” (Cat No. AF-107-0040). hMeDIP was performed with 3.5 μg of the rabbit 5-hmC antibody and the IP’d DNA was analysed by qPCR using primers specific for the 3 different control sequences. Figure 1 shows that the Diagenode rabbit polyclonal antibody against 5-hmC is highly specific for the 5-hmC base modification (no IP with non-methylated or methylated C control fragments).


    Figure 2 Determination of the antibody titer
    To determine the titer, an ELISA was performed using a serial dilution of the Diagenode antibody directed against 5-hmC (cat. No. pAb-HMC-050), crude serum and flow through, in antigen coated wells. The antigen used was the 5-hmC base coupled to BSA. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:2,800.

    Dot blot

    Figure 3 Dot blot analysis using the Diagenode antibody directed against 5-hmC
    To demonstrate the specificity of the Diagenode antibody against 5-hmC (cat. No. pAb-HMC-050), a Dot blot analysis was performed using the hmC, mC and C controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat No. AF-101-0002). One hundred to 4 ng (equivalent of 5 to 0.2 pmol of C-bases) of the controls were spotted on a membrane (Amersham Hybond-N+). The antibody was used at a dilution of 1:1,000. Figure 3 shows a high specificity of the antibody for the hydroxymethylated control.

  • Target description

    5-hydroxymethylcytosine (5-hmC) has been recently discovered in mammalian DNA. This results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. So far, the 5-hmC bases have been identified in Purkinje neurons, in granule cells and embryonic stem cells where they are present at high levels (up to 0,6% of total nucleotides in Purkinje cells).

    Preliminary results indicate that 5-hmC may have important roles distinct from 5-mC. Although its precise role has still to be shown, early evidence suggests a few putative mechanisms that could have big implications in epigenetics : 5-hydroxymethylcytosine may well represent a new pathway to demethylate DNA involving a repair mechanism converting 5-hmC to cytosine and, as such open up entirely new perspectives in epigenetic studies.

    Due to the structural similarity between 5-mC and 5-hmC, these bases are experimentally almost indistinguishable. Recent articles demonstrated that the most common approaches (e.g. enzymatic approaches, bisulfite sequencing) do not account for 5-hmC. The development of the affinity-based technologies appears to be the most powerful way to differentially and specifically enrich 5-mC and 5-hmC sequences. The results shown here illustrate the use of this unique monoclonal antibody against 5-hydroxymethylcytosine that has been fully validated in various technologies.

  •  Applications
    Methylated DNA immunoprecipitation
    Sensitive tumour detection and classification using plasma cell-free DNA methylomesRead the publication Preparation of cfMeDIP-seq libraries for methylome profiling of plasma cell-free DNARead the method The Methylated DNA Immunoprec... Read more
    Enzyme-linked immunosorbent assay. Read more
    Dot blotting Read more
  •  Documents
    Datasheet 5hmC pAb-HMC-050 DATASHEET
    Polyclonal antibody raised in rabbit against 5-hydroxymethylcytosine conjugated to KLH.
    Exclusive Highly Specific Kits Antibodies for DNA HydroxyMethylation Studies POSTER
    Cytosine hydroxymethylation was recently discovered as an important epigenetic mechanism. This cy...
    Epigenetic Antibodies Brochure BROCHURE
    More than in any other immuoprecipitation assays, quality antibodies are critical tools in many e...
    Antibodies you can trust POSTER
    Epigenetic research tools have evolved over time from endpoint PCR to qPCR to the analyses of lar...
  •  Safety sheets
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  •  Publications

    How to properly cite this product in your work

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    DNA methylation dynamics underlie metamorphic gene regulation programs in Xenopus tadpole brain.
    Kyono Y, Raj S, Sifuentes CJ, Buisine N, Sachs L, Denver RJ
    Methylation of cytosine residues in DNA influences chromatin structure and gene transcription, and its regulation is crucial for brain development. There is mounting evidence that DNA methylation can be modulated by hormone signaling. We analyzed genome-wide changes in DNA methylation and their relationship to gene ...

    5-hydroxymethylcytosine marks postmitotic neural cells in the adult and developing vertebrate central nervous system
    Diotel N et al.
    The epigenetic mark 5-hydroxymethylcytosine (5hmC) is a cytosine modification that is abundant in the central nervous system of mammals and which results from 5-methylcytosine oxidation by TET enzymes. Such a mark is suggested to play key roles in the regulation of chromatin structure and gene expression. However, i...

    Transcriptome-wide mapping of 5-methylcytidine RNA modifications in bacteria, archaea, and yeast reveals m5C within archaeal mRNAs.
    Edelheit S, Schwartz S, Mumbach MR, Wurtzel O, Sorek R
    The presence of 5-methylcytidine (m(5)C) in tRNA and rRNA molecules of a wide variety of organisms was first observed more than 40 years ago. However, detection of this modification was limited to specific, abundant, RNA species, due to the usage of low-throughput methods. To obtain a high resolution, systematic, an...

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