5-hydroxymethylcytosine (5-hmC) Antibody (rat)
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.
| Lot | 002 |
|---|---|
| Concentration | 1 µg/µl |
| Species reactivity | Human, mouse, other (wide range) |
| Type | Monoclonal |
| Purity | Affinity purified |
| Host | Rat |
| Precautions | This 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 |
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C02010034Auto hMeDIP kit x16 (monoclonal mouse antibody)
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C02010031
hMeDIP kit x16 (monoclonal mouse antibody)
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C15410255-100TET2 Antibody
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C15410311TET3 Antibody
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C15200081-100
5-methylcytosine (5-mC) Antibody - clone 33D3
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5-Carboxylcytosine (5-caC) Antibody
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5-formylcytosine (5-fC) Antibody
Validation Data
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.
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.
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.
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 Cytosine hydroxymethylation was recently discovered as an important epigenetic mechanism. This cy... | Download |
| Antibodies you can trust POSTER Epigenetic research tools have evolved over time from endpoint PCR to qPCR to the analyses of lar... | Download |
Safety sheets
Publications
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Anticancer Activity of Jania rubens in HCT-116 Cells via EMT Suppression, TET Downregulation, and ROS-Mediated Cytotoxicity |
DNMT1 regulates the timing of DNA methylation by DNMT3 in anenzymatic activity-dependent manner in mouse embryonic stem cells. |
Functional role of Tet-mediated RNA hydroxymethylcytosine in mouse ES
cells and during differentiation. |
Global distribution of DNA hydroxymethylation and DNA methylation in chronic lymphocytic leukemia. |
Regulation of the DNA Methylation Landscape in Human Somatic Cell Reprogramming by the miR-29 Family |
Transcriptome-wide distribution and function of RNA hydroxymethylcytosine |
RNA biochemistry. Transcriptome-wide distribution and function of RNA hydroxymethylcytosine. |
CpG signalling, H2A.Z/H3 acetylation and microRNA-mediated deferred self-attenuation orchestrate foetal NOS3 expression. |
White matter tract and glial-associated changes in 5-hydroxymethylcytosine following chronic cerebral hypoperfusion. |
Tet2 Facilitates the Derepression of Myeloid Target Genes during CEBPα-Induced Transdifferentiation of Pre-B Cells. |
Lineage-specific distribution of high levels of genomic 5-hydroxymethylcytosine in mammalian development. |
Genome-wide analysis of 5-hydroxymethylcytosine distribution reveals its dual function in transcriptional regulation in mouse embryonic stem cells. |