Diagenode

5-hydroxymethylcytosine (5-hmC) polyclonal antibody (rabbit) - Classic

RFX5-polyclonal-antibody-diagenode
Catalog Number
Format
Price
C15310210-20
20 µl
$90.00
  Bulk order
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Polyclonal antibody raised against 5-hydroxymethylcytosine (5-hmC). 5-hmC is a recently discovered DNA modification which results from the enzymatic conversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET family of oxygenases. Preliminary results indicate that 5-hmC may have important roles distinct from 5-methylcytosine (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.

LotA1204-004
Concentrationnot determined
Species reactivityHuman, mouse, other (wide range)
TypePolyclonal
PurityWhole antiserum
HostRabbit
PrecautionsThis product is for research use only. Not for use in diagnostic or therapeutic procedures.
Applications Suggested dilution References
ELISA 1/500 Fig 1
hMeDIP 2.5 μl/IP Fig 2
Dot Blotting 1:200 Fig 3
  • Validation Data

    ELISA

    Figure 1. Determination of the 5-hmC rabbit polyclonal antibody titer
    To determine the titer, an ELISA was performed using a serial dilution of the Diagenode rabbit polyclonal antibody directed against 5-hmC in antigen coated wells. The antigen used was BSA coupled to the 5-hmC base. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1: 3,500.

    Figure 2. An hydroxymethylated DNA IP (hMeDIP) was performed using the Diagenode rabbit polyclonal antibody directed against 5-hydroxymethylcytosine (Cat. No. CS-HMC-100).
    The IgG isotype antibodies from rabbit (Cat. No. kch-504-250) was used as negative control. The DNA was prepared with the GenDNA module of the hMeDIP kit and sonicated with our Bioruptor® (UCD-200/300 series) to have DNA fragments of 300-500 bp. 1 μg of human Hela cells DNA were spiked with non-methylated, methylated, and hydroxymethylated fragments. The IP’d material has been analysed by qPCR using the primer pair specific for the 3 different control sequences. The obtained results show that the Diagenode rabbit polyclonal for 5-hmC is highly specific for this base modification (no IP with non-methylated or methylated C bases containing fragments).

    Dot Blot

    Figure 3. Dotblot analysis of the Diagenode 5-hmC rabbit polyclonal antibody with the C, mC and hmC PCR controls
    100 to 4 ng (equivalent of 5 to 0.2 pmol of C-bases) of the hmC, mC and C PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with the rabbit 5-hydroxymethylcytosine polyclonal antibody (dilution 1:200). The membranes were exposed for 30 seconds.

  • 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
    The Methylated DNA IP (MeDIP) is based on the affinity purification of methylated DNA using an antibody directed against 5-methylcytosine (5-mC) or 5-hydroxymethylcytosine (5-hmC) in the case of hMeDIP. How it works In brief, Methyl DNA IP is ... Read more
    ELISA
    Enzyme-linked immunosorbent assay. Read more
    DB
    Dot blotting Read more
  • Documents
    Exclusive Highly Specific Kits Antibodies for DNA HydroxyMethylation Studies POSTER
    5-hydroxymethylcytosine (5-hmC) has been recently discovered in mammalian DNA by two US groups (K...
    Download
    Datasheet 5hmC CS-HMC-050 DATASHEET
    Polyclonal antibody raised in rabbit against 5-hydroxymethylcytosine conjugated to KLH.
    Download
  • Publications

    How to properly cite this product in your work

    Diagenode strongly recommends using this: 5-hydroxymethylcytosine (5-hmC) polyclonal antibody (rabbit) - Classic (Diagenode Cat# C15310210-20 Lot# A1204-004). Click here to copy to clipboard.

    Using our products in your publication? Let us know!

    RESEARCH RESOURCE: Changes in gene expression and Estrogen Receptor cistrome in mouse liver upon acute E2 treatment.
    Palierne G et al.
    Transcriptional regulation by the Estrogen Receptor α (ER) has been investigated mainly in breast cancer cell lines but estrogens such as 17β-Estradiol (E2) exert numerous extra-reproductive effects, particularly in the liver where E2 exhibits both protective metabolic and deleterious thrombotic actions. ...

    Peroxisome proliferator-activated receptor γ and C/EBPα synergistically activate key metabolic adipocyte genes by assisted loading.
    Madsen MS, Siersbæk R, Boergesen M, Nielsen R, Mandrup S
    Peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) are key activators of adipogenesis. They mutually induce the expression of each other and have been reported to cooperate in activation of a few adipocyte genes. Recently, genome-wide profiling ...

    Dynamic hydroxymethylation of deoxyribonucleic acid marks differentiation-associated enhancers.
    Sérandour AA, Avner S, Oger F, Bizot M, Percevault F, Lucchetti-Miganeh C, Palierne G, Gheeraert C, Barloy-Hubler F, Péron CL, Madigou T, Durand E, Froguel P, Staels B, Lefebvre P, Métivier R, Eeckhoute J, Salbert G
    Enhancers are developmentally controlled transcriptional regulatory regions whose activities are modulated through histone modifications or histone variant deposition. In this study, we show by genome-wide mapping that the newly discovered deoxyribonucleic acid (DNA) modification 5-hydroxymethylcytosine (5hmC) is dy...

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