Diagenode

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

RFX5-polyclonal-antibody-diagenode
Catalog Number
Format
Price
C15220001-50
(MAb-633HMC-050)
50 µg/32 µl
$295.00
  Bulk order
Other format

5-hmC is a 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.

LotIgG2a
Concentration1.6 µg/µl
Species reactivityHuman, mouse, other (wide range)
TypeMonoclonal
PurityAffinity purified
HostRat
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 1:1,000 Fig 2
Dot Blotting 1:500 (4 μg/ml) Fig 3, 4
  • Validation Data

    hMeDIP

    Figure 1. Hydroxymethylated DNA IP results obtained with our hMeDIP kit (Cat. No. AF-104-0016)
    Hydroxymethylated DNA IP (hMeDIP) assays were performed using the Diagenode hMeDIP kit. This kit includes: the monoclonal antibody against 5-hydroxymethylcytosine (Cat. No. MAb-633HMC-050), 5-hmC, 5-mC & cytosine DNA standards & Rat IgG (Cat. No. AF-105-0025). The DNA was prepared with the GenDNA module and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. 1 μg of mouse ES cells DNA was spiked with 0.025 ng of each DNA standard. The IP’d material has been analysed by qPCR using the primer pairs specific to the control sequences. The obtained results are as follows: - hMeDIP on unmethylated control • with Rat IgG as negative control (0.06%, almost no recovery) • with 5-hmC antibody (0.61%, almost no recovery) - hMeDIP on methylated control • with Rat IgG as negative control (0.03%, almost no recovery) • with 5-hmC antibody (0.62%, almost no recovery) - hMeDIP on hydroxymethylated control • with Rat IgG as negative control (0.04%, almost no recovery) • with 5-hmC (97.60% recovery, almost full recovery) These results clearly demonstrate the high specificity and efficiency of the 5-hydroxymethylcytosine monoclonal antibody.

    ELISA

    Figure 2. Determination of the 5-hmC rat monoclonal antibody titer
    To determine the titer, an ELISA was performed using a serial dilution of the Diagenode monoclonal antibody directed against 5-hmC (Cat No. MAb-633HMC-050, MAb-633HMC-100) in antigen coated wells. The antigen used was a 5-hmC base coupled to KHL. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:25,000.

    Dot blot

    Figure 3. Dot blot analysis of the Diagenode 5-hmC and 5-mC monoclonal antibodies with the C, mC and hmC PCR controls
    Figure 3A: Approximately 200 ng, equivalent 10 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-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 5-hydroxymethylcytosine rat monoclonal antibody (dilution 1:500 ; 4 μg/ml final concentration), followed by an HRP conjugated anti-rat secondary antibody. The membrane was exposed during 30 seconds. Figure 3B: Incubation of the same membrane with the 5-methylcytosine mouse monoclonal antibody (Cat. No. MAb-335MEC-100/500) (dilution 1:250). Note that the membrane was not stripped after the 5-hmC incubation. The left spot represents the remaining hmC signal. This result confirms that an equal amount of mC bases was spotted at position 2.

    Figure 4. Dot blot analysis of the Diagenode 5-hmC rat monoclonal antibody with the C, mC and hmC PCR controls
    200 to 2 ng (equivalent of 10 to 0.1 pmol of C-base) 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 4 μg/ml (dilution 1:500) of the 5-hydroxymethylcytosine rat monoclonal antibody, followed by an HRP conjugated anti-rat secondary antibody. The membrane was 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.

  • Protocols
  • 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-633HMC-100 DATASHEET
    5-hmC is a DNA modification which results from the enzymatic conversion of 5-methylcytosine into ...
    Download
    Epigenetic Antibodies Brochure BROCHURE
    More than in any other immuoprecipitation assays, quality antibodies are critical tools in many e...
    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
    Antibodies you can trust POSTER
    Epigenetic research tools have evolved over time from endpoint PCR to qPCR to the analyses of lar...
    Download
  • Publications

    How to properly cite this product in your work

    Diagenode strongly recommends using this: 5-hydroxymethylcytosine (5-hmC) monoclonal antibody (rat) - Classic (Diagenode Cat# C15220001-50 Lot# IgG2a). Click here to copy to clipboard.

    Using our products in your publication? Let us know!

    Regulation of the DNA Methylation Landscape in Human Somatic Cell Reprogramming by the miR-29 Family
    Hysolli E et al.
    Reprogramming to pluripotency after overexpression of OCT4, SOX2, KLF4, and MYC is accompanied by global genomic and epigenomic changes. Histone modification and DNA methylation states in induced pluripotent stem cells (iPSCs) have been shown to be highly similar to embryonic stem cells (ESCs). However, epigenetic d...

    Transcriptome-wide distribution and function of RNA hydroxymethylcytosine
    Delatte B, Wang F, Ngoc LV, Collignon E, Bonvin E, Deplus R, Calonne E, Hassabi B, Putmans P, Awe S, Wetzel C, Kreher J, Soin R, Creppe C, Limbach PA, Gueydan C, Kruys V, Brehm A, Minakhina S, Defrance M, Steward R, Fuks F.
    Hydroxymethylcytosine, well described in DNA, occurs also in RNA. Here, we show that hydroxymethylcytosine preferentially marks polyadenylated RNAs and is deposited by Tet in Drosophila. We map the transcriptome-wide hydroxymethylation landscape, revealing hydroxymethylcytosine in the transcripts of many genes, nota...

    CpG signalling, H2A.Z/H3 acetylation and microRNA-mediated deferred self-attenuation orchestrate foetal NOS3 expression.
    Postberg J, Kanders M, Forcob S, Willems R, Orth V, Hensel KO, Weil PP, Wirth S, Jenke AC
    BACKGROUND: An adverse intrauterine environment leads to permanent physiological changes including vascular tone regulation, potentially influencing the risk for adult vascular diseases. We therefore aimed to monitor responsive NOS3 expression in human umbilical artery endothelial cells (HUAEC) and to study the unde...

    White matter tract and glial-associated changes in 5-hydroxymethylcytosine following chronic cerebral hypoperfusion.
    Tsenkina Y, Ruzov A, Gliddon C, Horsburgh K, De Sousa PA
    White matter abnormalities due to age-related cerebrovascular alterations is a common pathological hallmark associated with functional impairment in the elderly which has been modeled in chronically hypoperfused mice. 5-Methylcytosine (5mC) and its oxidized derivative 5-hydroxymethylcytosine (5hmC) are DNA modificat...

    Tet2 Facilitates the Derepression of Myeloid Target Genes during CEBPα-Induced Transdifferentiation of Pre-B Cells.
    Kallin EM, Rodríguez-Ubreva J, Christensen J, Cimmino L, Aifantis I, Helin K, Ballestar E, Graf T
    The methylcytosine hydroxylase Tet2 has been implicated in hematopoietic differentiation and the formation of myeloid malignancies when mutated. An ideal system to study the role of Tet2 in myelopoeisis is CEBPα-induced transdifferentiation of pre-B cells into macrophages. Here we found that CEBPα binds to upstream ...

    Lineage-specific distribution of high levels of genomic 5-hydroxymethylcytosine in mammalian development.
    Ruzov A, Tsenkina Y, Serio A, Dudnakova T, Fletcher J, Bai Y, Chebotareva T, Pells S, Hannoun Z, Sullivan G, Chandran S, Hay DC, Bradley M, Wilmut I, De Sousa P
    Methylation of cytosine is a DNA modification associated with gene repression. Recently, a novel cytosine modification, 5-hydroxymethylcytosine (5-hmC) has been discovered. Here we examine 5-hmC distribution during mammalian development and in cellular systems, and show that the developmental dynamics of 5-hmC are d...

    Genome-wide analysis of 5-hydroxymethylcytosine distribution reveals its dual function in transcriptional regulation in mouse embryonic stem cells.
    Wu H, D'Alessio AC, Ito S, Wang Z, Cui K, Zhao K, Sun YE, Zhang Y
    Recent studies have demonstrated that the Ten-eleven translocation (Tet) family proteins can enzymatically convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). While 5mC has been studied extensively, little is known about the distribution and function of 5hmC. Here we present a genome-wide profile of 5h...

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