5-Carboxylcytosine (5-caC) Antibody
| Lot | 001 |
|---|---|
| Concentration | 1.0 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 |
|---|---|---|
| Dot Blotting | 1:500 - 1:1,000 | Fig 1 |
| Immunofluorescence | 1:500 | Fig 2 |
| IP * | 4 μg/IP (4 μg genomic DNA per IP) | Fig 3 |
* Please note that of the optimal antibody amount per IP should be determined by the end-user. We recommend testing 1-5 μg per IP.
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Validation Data
Fig. 1. Dot blot analysis using the Diagenode antibody directed against 5-caC
To demonstrate the specificity of the Diagenode antibody against 5-caC (cat. No. pAb-CaC-020/050), a Dot Blot analysis was performed using synthetic oligonucleotides containing different modified C-bases (indicated in red). 125 and 25 ng of the respective oligo’s were bound to a Streptavindin-coated multi-well plate. The antibody was used at a dilution of 1:1,000. The binding of antibody to the DNA was measured by ECL chemiluminescence. Figure 1 shows a high specificity of the antibody for the carboxylated cytosine.
Fig. 2. Immunofluorescence assay using the Diagenode antibody directed against 5-caC
293T cells were transfected with either the mouse FLAG-tagged wild-type Tet1 (Tet1 CD) or the catalytically inactive FLAG-tagged C-terminal domain of 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.
Fig. 3. 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.
Target description
Until recently, 5-methylcytosine (5-mC) was the only known modification of DNA for epigenetic regulation. In 2009, however, a second methylated cytosine, 5-hydroxymethylcytosine (5-hmC) was discovered. This new modified base (also called the Sixth base) is generated by enzymatic conversion of 5-mC into 5-hmC by the TET family of oxygenases.
Recent results indicate that 5-hmC plays important roles distinct from 5-mC. Although its precise role has still to be shown, early evidence suggests that 5-hmC may well represent a new pathway to demethylate DNA involving a repair mechanism converting 5-hmC to cytosine. This pathway 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, the different 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 and 5-hmC sequences. We previously released highly specific antibodies directed against 5-mC and 5-hmC. Now, we also present a unique rabbit polyclonal antibody against 5-Carboxycytosine.
Applications
| DB Dot blotting Read more |
| IF Immunofluorescence: Diagenode offers huge selection of highly sensitive antibodies validated in IF. Immunofluorescence using the Diagenode monoclonal antibody directed against CRISPR/Cas9 HeLa cells transfected with a Cas9 expression vector (... Read more |
| IP Immunoprecipitation Read more |
Documents
| Datasheet 5-caC pAb-cac-100 DATASHEET Polyclonal antibody raised in rabbit against 5-Carboxylcytosine (5ca-CMP monophosphate) conjugate... | Download |
| Antibodies you can trust POSTER Epigenetic research tools have evolved over time from endpoint PCR to qPCR to the analyses of lar... | Download |
| Epigenetic Antibodies Brochure BROCHURE More than in any other immuoprecipitation assays, quality antibodies are critical tools in many e... | Download |
Safety sheets
| SDS C15410204 5-caC Antibody US en | Download |
| SDS C15410204 5-caC Antibody GB en | Download |
| SDS C15410204 5-caC Antibody DE de | Download |
| SDS C15410204 5-caC Antibody JP ja | Download |
| SDS C15410204 5-caC Antibody ES es | Download |
| SDS C15410204 5-caC Antibody FR fr | Download |
| SDS C15410204 5-caC Antibody BE fr | Download |
| SDS C15410204 5-caC Antibody BE nl | Download |
Publications
How to properly cite our product/service in your workWe strongly recommend using this: 5-Carboxylcytosine (5-caC) Antibody (Hologic Diagenode Cat# C15410204-100 Lot# 001). Click here to copy to clipboard. Using our products or services in your publication? Let us know! |
A transition phase in late mouse oogenesis impacts DNA methylation ofthe early embryo. |
Regulation of paternal 5mC oxidation and H3K9me2 asymmetry byERK1/2 in mouse zygotes. |
DNA methylation dynamics underlie metamorphic gene regulation programs in Xenopus tadpole brain. |
Sodium valproate and 5-aza-2'-deoxycytidine differentially modulate DNA demethylation in G1 phase-arrested and proliferative HeLa cells. |
Active and passive demethylation of male and female pronuclear DNA in the Mammalian zygote. |