5-hydroxymethylcytosine (5-hmC) Monoclonal Antibody (rat)

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

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

Dot blot protocol for 5-hydroxymethylcytosine monoclonal rat antibody

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Applications

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 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

5-hmC monoclonal antibody rat SDS US en	Download
5-hmC monoclonal antibody rat SDS GB en	Download
5-hmC monoclonal antibody rat SDS BE fr	Download
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5-hmC monoclonal antibody rat SDS ES es	Download
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5-hmC monoclonal antibody rat SDS BE nl	Download

Publications

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DNMT1 regulates the timing of DNA methylation by DNMT3 in anenzymatic activity-dependent manner in mouse embryonic stem cells.
Ito Takamasa et al.
DNA methylation (DNAme; 5-methylcytosine, 5mC) plays an essential role in mammalian development, and the 5mC profile is regulated by a balance of opposing enzymatic activities: DNA methyltransferases (DNMTs) and Ten-eleven translocation dioxygenases (TETs). In mouse embryonic stem cells (ESCs), de novo DNAme by DNMT...

Functional role of Tet-mediated RNA hydroxymethylcytosine in mouse ES cells and during differentiation.
Lan, Jie and Rajan, Nicholas and Bizet, Martin and Penning, Audrey and Singh, Nitesh K and Guallar, Diana and Calonne, Emilie and Li Greci, Andrea and Bonvin, Elise and Deplus, Rachel and Hsu, Phillip J and Nachtergaele, Sigrid and Ma, Chengjie and Song,
Tet-enzyme-mediated 5-hydroxymethylation of cytosines in DNA plays a crucial role in mouse embryonic stem cells (ESCs). In RNA also, 5-hydroxymethylcytosine (5hmC) has recently been evidenced, but its physiological roles are still largely unknown. Here we show the contribution and function of this mark in mouse ESCs...

Global distribution of DNA hydroxymethylation and DNA methylation in chronic lymphocytic leukemia.
Wernig-Zorc S, Yadav MP, Kopparapu PK, Bemark M, Kristjansdottir HL, Andersson PO, Kanduri C, Kanduri M
BACKGROUND: Chronic lymphocytic leukemia (CLL) has been a good model system to understand the functional role of 5-methylcytosine (5-mC) in cancer progression. More recently, an oxidized form of 5-mC, 5-hydroxymethylcytosine (5-hmC) has gained lot of attention as a regulatory epigenetic modification with prognostic ...

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...

RNA biochemistry. 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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