Diagenode

5-hydroxymethylcytosine (5-hmC) monoclonal antibody (mouse) - Classic

RFX5-polyclonal-antibody-diagenode
Catalog Number
Format
Price
C15200200-100
(Mab-31HMC-100)
100 µg/100 µl
$395.00
  Bulk order
Other format

One of the only two monoclonal antibodies 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.

Lot001
Concentration1.0 µg/µl
Species reactivityHuman, mouse, other (wide range)
TypeMonoclonal
PurityProtein G purified
HostMouse
PrecautionsThis product is for research use only. Not for use in diagnostic or therapeutic procedures.
Applications Suggested dilution References
hMeDIP 2.5 μg/IP Fig 1
ELISA 1:500 Fig 2
Dot Blotting 2 μg / ml Fig 3
  • Validation Data

    ChIP

    Figure 1. An hydroxymethylated DNA IP (hMeDIP) was performed using the Diagenode mouse monoclonal antibody directed against 5-hydroxymethylcytosine (Cat. No. MAb-31HMC-020, MAb-31HMC-050, MAb-31HMC-100).
    The IgG isotype antibodies from mouse (Cat. No. kch-819-015) 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 PCR 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 mouse monoclonal for 5-hmC is highly specific for this base modification (no IP with non-methylated or methylated C bases containing fragments).

    ELISA

    Figure 2. Determination of the 5-hmC mouse monoclonal antibody titer
    To determine the titer, an ELISA was performed using a serial dilution of the Diagenode mouse monoclonal antibody directed against 5-hmC (Cat No. MAb-31HMC-050, MAb-31HMC-100) in antigen coated wells. The antigen used was KHL coupled to 5-hmC base. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:40,000.

    Dot Blot

    Figure 3. Dotblot analysis of the Diagenode 5-hmC mouse monoclonal antibody with the C, mC and hmC PCR controls
    200 to 2 ng (equivalent of 10 to 0.1 pmol of C-bases) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 2 μg/ml of the mouse 5-hydroxymethylcytosine monoclonal antibody (dilution 1:500). 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
    Epigenetics DNA Methylation
    Complete solutions for DNA methylation studies Whether you are experienced or new to the field of DNA methylation, Diagenode has everything you need to make your assay as easy and convenient as possible while ensuring consistent data betwee... Read more
    DB
    Dot blotting Read more
    ELISA
    Enzyme-linked immunosorbent assay. Read more
  • Documents
    Datasheet 5hmC MAb-31HMC-050 DATASHEET
    Monoclonal antibody raised in mouse against 5-hydroxymethylcytosine conjugated to BSA.
    Download
    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
  • Publications

    How to properly cite this product in your work

    Diagenode strongly recommends using this: 5-hydroxymethylcytosine (5-hmC) monoclonal antibody (mouse) - Classic (Diagenode Cat# C15200200-100 Lot# 001). Click here to copy to clipboard.

    Using our products in your publication? Let us know!

    Dynamic interplay between locus-specific DNA methylation and hydroxymethylation regulates distinct biological pathways in prostate carcinogenesis
    Kamdar SN, Ho LT, Kron KJ, Isserlin R, van der Kwast T, Zlotta AR, Fleshner NE, Bader G, Bapat B
    Background Despite the significant global loss of DNA hydroxymethylation marks in prostate cancer tissues, the locus-specific role of hydroxymethylation in prostate tumorigenesis is unknown. We characterized hydroxymethylation and methylation marks by performing whole-genome next-generation sequencing in represen...

    Hydroxymethylation of microRNA-365-3p Regulates Nociceptive Behaviors via Kcnh2
    Pan Z, Zhang M, Ma T, Xue Z-Y, Li G-F, Hao L-Y, Zhu L-J, Li Y-Q, Ding H-L, Cao J-L
    DNA 5-hydroxylmethylcytosine (5hmC) catalyzed by ten-eleven translocation methylcytosine dioxygenase (TET) occurs abundantly in neurons of mammals. However, the in vivo causal link between TET dysregulation and nociceptive modulation has not been established. Here, we found that spinal TET1 and TET3 were significant...

    The dual specificity phosphatase 2 gene is hypermethylated in human cancer and regulated by epigenetic mechanisms
    Tanja Haag, Antje M. Richter, Martin B. Schneider, Adriana P. Jiménez and Reinhard H. Dammann
    Dual specificity phosphatases are a class of tumor-associated proteins involved in the negative regulation of the MAP kinase pathway. Downregulation of the dual specificity phosphatase 2 (DUSP2) has been reported in cancer. Epigenetic silencing of tumor suppressor genes by abnormal promoter methylation is ...

    Single-Base Resolution Analysis of 5-Formyl and 5-Carboxyl Cytosine Reveals Promoter DNA Methylation Dynamics.
    Neri F, Incarnato D, Krepelova A, Rapelli S, Anselmi F, Parlato C, Medana C, Dal Bello F, Oliviero S
    Ten eleven translocation (Tet) proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC can be further excised by thymine-DNA glycosylase (Tdg). Here, we present a genome-wide approach, named methylation-assisted bisulfite seq...

    Dynamic reprogramming of 5-hydroxymethylcytosine during early porcine embryogenesis.
    Cao Z, Zhou N, Zhang Y, Zhang Y, Wu R, Li Y, Zhang Y, Li N
    DNA active demethylation is an important epigenetic phenomenon observed in porcine zygotes, yet its molecular origins are unknown. Our results show that 5-methylcytosine (5mC) converts into 5-hydroxymethylcytosine (5hmC) during the first cell cycle in porcine in vivo fertilization (IVV), IVF, and SCNT embryos, but n...

    Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells.
    Blaschke K, Ebata KT, Karimi MM, Zepeda-Martínez JA, Goyal P, Mahapatra S, Tam A, Laird DJ, Hirst M, Rao A, Lorincz MC, Ramalho-Santos M
    DNA methylation is a heritable epigenetic modification involved in gene silencing, imprinting, and the suppression of retrotransposons. Global DNA demethylation occurs in the early embryo and the germ line, and may be mediated by Tet (ten eleven translocation) enzymes, which convert 5-methylcytosine (5mC) to 5-hydro...

    5-Hydroxymethylcytosine is associated with enhancers and gene bodies in human embryonic stem cells.
    Stroud H, Feng S, Morey Kinney S, Pradhan S, Jacobsen SE
    BACKGROUND: 5-Hydroxymethylcytosine (5hmC) was recently found to be abundantly present in certain cell types, including embryonic stem cells. There is growing evidence that TET proteins, which convert 5-methylcytosine (5mC) to 5hmC, play important biological roles. To further understand the function of 5hmC, an anal...

    5-Hydroxymethylcytosine in the mammalian zygote is linked with epigenetic reprogramming.
    Wossidlo M, Nakamura T, Lepikhov K, Marques CJ, Zakhartchenko V, Boiani M, Arand J, Nakano T, Reik W, Walter J
    The epigenomes of early mammalian embryos are extensively reprogrammed to acquire a totipotent developmental potential. A major initial event in this reprogramming is the active loss/demethylation of 5-methylcytosine (5mC) in the zygote. Here, we report on findings that link this active demethylation to molecular me...

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