Most throughly validated
5-carboxylcytosine antibody

 

Validated for IF & DNA Immunoprecipitation

5-Carboxylcytosine-Validation data
5-Carboxylcytosine-Immunofluorescence Assay

Immunofluorescence assay using the Diagenode antibody directed against 5-caC
293T cells were transfected with the FLAG-tagged C-terminal domain of either wild-type Tet1 (Tet1 CD) or catalytically inactive Tet1 (Tet1 mCD) and stained with the Diagenode antibody against 5-caC (Cat. No. pAb- CaC-020/050), diluted 1:500, and with an anti-FLAG antibody, followed by DAPI counterstaining.

5-Carboxylcytosine-Immunoprecipitation Graph

Immunoprecipitation using the Diagenode antibody directed against 5-caC
Immunoprecipitation was performed with the Diagenode antibody against 5-caC (cat. No. pAb-CaC-020/050) on 2 µg of J1 ES genomic DNA, spiked with 1 pg of a control DNA fragment (approximately 700 bp from the RFP (Ring finger protein) gene) containing different cytosine modifications. The mC and hmC control DNA was generated by PCR with the corresponding nucleotide. The caC control fragment was obtained by in vitro methylation using M.SssI methyltransferase followed by oxidation with purified Tet2. The IP'd DNA was subsequently anaysed by qPCR using primers specific for the control DNA fragments and for GAPDH, used as a negative control. Figure 3 shows the enrichment calculated as the ratio of the recovery of the control DNA versus the recovery of the GAPDH negative control.

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Recent progress in epigenetics demonstrates that the Tet family catalyzes in vivo conversion of:

  • 5-methylcytosine (5-mC) to
    5-hydroxymethylcytosine (5-hmC)
  • 5-hmC to 5-formylcytosine (5-fC)
    and 5-carboxylcytosine (5-caC)

Until recently, 5-methylcytosine (5-mC) was the only known modification of DNA for epigenetic regulation. In 2009, however, Kriaucionis1 discovered a second methylated cytosine, 5-hydroxymethylcytosine (5-hmC). The so-called 6th base, is generated by enzymatic conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine by the TET family of oxygenases.

Early reports suggested that 5-hmC may represent an intermediate of active demethylation in a new pathway which demethylates DNA, converting 5-mC to cytosine. Recent evidence fuel the hypothesis that 5-hmC represents an intermediate, which could involve further oxidation of the hydroxymethyl group to a formyl or carboxyl group followed by either deformylation or decarboxylation. The carboxyl and formyl groups of 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) could be enzymatically removed without excision of the base.

Due to their structural similarity, these modified cytosine analogues are difficult to discriminate. The development of highly specific affinity-based reagents (such as antibodies) appears to be the most powerful way to differentially and specifically enrich 5-mC, 5-hmC & 5-fC sequences. In this context Diagenode is proud to offer a validated antibody against
5-caC.

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