Diagenode

5-hydroxymethylcytosine (5-hmC) polyclonal antibody (rabbit) - Classic

カタログ番号
フォーマット
価格
C15410205-20
(pAb-HMC-020)
20 µg
$100.00
  Bulk order
ブローカー情報

Polyclonal antibody 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.

LotA1203-0041
Concentration3.5 µg/µl
Species reactivityHuman, mouse, other (wide range)
TypePolyclonal
PurityAffinity purified
HostRabbit
PrecautionsThis 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
  • Validation Data

    hMeDIP

    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).

    ELISA

    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.

    Dot blot

    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.

  •  実験手法
    DB
    Dot blotting Read more
    ELISA
    Enzyme-linked immunosorbent assay. Read more
  •  資料
    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...
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    Datasheet 5hmC pAb-HMC-050 DATASHEET
    Polyclonal antibody raised in rabbit against 5-hydroxymethylcytosine conjugated to KLH.
    Download
  •  出版物

    How to properly cite this product in your work

    Diagenode strongly recommends using this: 5-hydroxymethylcytosine (5-hmC) polyclonal antibody (rabbit) - Classic (Diagenode Cat# C15410205-20 Lot# A1203-0041). Click here to copy to clipboard.

    Using our products in your publication? Let us know!

    5-hydroxymethylcytosine marks postmitotic neural cells in the adult and developing vertebrate central nervous system
    Diotel N et al.
    The epigenetic mark 5-hydroxymethylcytosine (5hmC) is a cytosine modification that is abundant in the central nervous system of mammals and which results from 5-methylcytosine oxidation by TET enzymes. Such a mark is suggested to play key roles in the regulation of chromatin structure and gene expression. However, i...

    Transcriptome-wide mapping of 5-methylcytidine RNA modifications in bacteria, archaea, and yeast reveals m5C within archaeal mRNAs.
    Edelheit S, Schwartz S, Mumbach MR, Wurtzel O, Sorek R
    The presence of 5-methylcytidine (m(5)C) in tRNA and rRNA molecules of a wide variety of organisms was first observed more than 40 years ago. However, detection of this modification was limited to specific, abundant, RNA species, due to the usage of low-throughput methods. To obtain a high resolution, systematic, an...

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