5-hydroxymethylcytosine (5-hmC) Antibody (rabbit)
Polyclonal antibody raised in rabbit against 5-hydroxymethylcytosine conjugated to KLH.
| Lot | A1203-0041 |
|---|---|
| Concentration | 3.5 µg/µl |
| Species reactivity | Human, mouse, other (wide range) |
| Type | Polyclonal |
| Purity | Affinity purified |
| Host | Rabbit |
| Precautions | This product is for research use only. Not for use in diagnostic or therapeutic procedures. |
| Applications | Suggested dilution | References |
|---|---|---|
| hMeDIP* | 3.5 μg/IP | Fig 1 |
| ELISA | 1:100 - 1:500 | Fig 2 |
| Dot Blotting | 1:1,1000 | Fig 3 |
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Validation Data
Figure 1 hMeDIP results obtained with the Diagenode antibody directed against 5-hmC
hMeDIP (hydroxymethylated DNA IP) was performed using the Diagenode antibody against 5-hydroxymethylcytosine (Cat. No. pAb-HMC-050). DNA from mouse ES cells was prepared with the GenDNA module of the hMeDIP kit and sonicated with our Bioruptor® (UCD-200/300 series) to obtain DNA fragments of 300-500 bp. One μg of sheared DNA was spiked with the unmethylated (C) methylated (mC), and hydroxymethylated (hmC) controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack for hMeDIP” (Cat No. AF-107-0040). hMeDIP was performed with 3.5 μg of the rabbit 5-hmC antibody and the IP’d DNA was analysed by qPCR using primers specific for the 3 different control sequences. Figure 1 shows that the Diagenode rabbit polyclonal antibody against 5-hmC is highly specific for the 5-hmC base modification (no IP with non-methylated or methylated C control fragments).
Figure 2 Determination of the antibody titer
To determine the titer, an ELISA was performed using a serial dilution of the Diagenode antibody directed against 5-hmC (cat. No. pAb-HMC-050), crude serum and flow through, in antigen coated wells. The antigen used was the 5-hmC base coupled to BSA. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:2,800.
Figure 3 Dot blot analysis using the Diagenode antibody directed against 5-hmC
To demonstrate the specificity of the Diagenode antibody against 5-hmC (cat. No. pAb-HMC-050), a Dot blot analysis was performed using the hmC, mC and C controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack” (Cat No. AF-101-0002). One hundred to 4 ng (equivalent of 5 to 0.2 pmol of C-bases) of the controls were spotted on a membrane (Amersham Hybond-N+). The antibody was used at a dilution of 1:1,000. Figure 3 shows a high specificity of the antibody for the hydroxymethylated control.
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 Sensitive tumour detection and classification using plasma cell-free DNA methylomesRead the publication Preparation of cfMeDIP-seq libraries for methylome profiling of plasma cell-free DNARead the method The Methylated DNA Immunoprec... Read more |
| ELISA Enzyme-linked immunosorbent assay. Read more |
| DB Dot blotting Read more |
Documents
| Datasheet 5hmC pAb-HMC-050 DATASHEET Polyclonal antibody raised in rabbit against 5-hydroxymethylcytosine conjugated to KLH. | Download |
| Exclusive Highly Specific Kits Antibodies for DNA HydroxyMethylation Studies POSTER Cytosine hydroxymethylation was recently discovered as an important epigenetic mechanism. This cy... | Download |
| Epigenetic Antibodies Brochure BROCHURE More than in any other immuoprecipitation assays, quality antibodies are critical tools in many e... | 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
| SDS C15410205 5-hydroxymethylcytosine 5-hmC Antibody rabbit US en | Download |
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| SDS C15410205 5-hydroxymethylcytosine 5-hmC Antibody rabbit FR fr | Download |
| SDS C15410205 5-hydroxymethylcytosine 5-hmC Antibody rabbit BE nl | Download |
| SDS C15410205 5-hydroxymethylcytosine 5-hmC Antibody rabbit BE fr | Download |
| SDS C15410205 5-hydroxymethylcytosine 5-hmC Antibody rabbit DE de | Download |
| SDS C15410205 5-hydroxymethylcytosine 5-hmC Antibody rabbit ES es | Download |
| SDS C15410205 5-hydroxymethylcytosine 5-hmC Antibody rabbit JP ja | Download |
Publications
How to properly cite our product/service in your workWe strongly recommend using this: 5-hydroxymethylcytosine (5-hmC) Antibody (rabbit) (Hologic Diagenode Cat# C15410205 Lot# A1203-0041). Click here to copy to clipboard. Using our products or services in your publication? Let us know! |
DNA methylation dynamics underlie metamorphic gene regulation programs in Xenopus tadpole brain. |
5-hydroxymethylcytosine marks postmitotic neural cells in the adult and developing vertebrate central nervous system |
Transcriptome-wide mapping of 5-methylcytidine RNA modifications in bacteria, archaea, and yeast reveals m5C within archaeal mRNAs. |