Diagenode

5-caC polyclonal antibody - Classic

カタログ番号
フォーマット
価格
C15410204-20
(pAb-caC-020)
20 µg
$100.00
  Bulk order
ブローカー情報

Diagenode, the leader in the field of epigenetics, provides full solutions for your chromatin immunoprecipitation and DNA methylation research. Our products include sonication instruments (Bioruptor®), automated systems (IP-Star®), kits for chromatin immunoprecipitation (ChIP) and DNA methylation analysis, and high quality antibodies. Our goal is to make epigenetics research accessible and to ensure consistent data in every experiment.

Lot001
Concentration1.0 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
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.

  • Validation Data

    Dot blot

    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.

    Immunofluorescence

    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.

    Immunoprecipitation

    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.

  •  実験手法
    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
  •  資料
    Datasheet 5-caC pAb-cac-100 DATASHEET
    Polyclonal antibody raised in rabbit against 5-Carboxylcytosine (5ca-CMP monophosphate) conjugate...
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  •  出版物

    How to properly cite this product in your work

    Diagenode strongly recommends using this: 5-caC polyclonal antibody - Classic (Diagenode Cat# C15410204-20 Lot# 001). Click here to copy to clipboard.

    Using our products in your publication? Let us know!

    Active and passive demethylation of male and female pronuclear DNA in the Mammalian zygote.
    Guo F, Li X, Liang D, Li T, Zhu P, Guo H, Wu X, Wen L, Gu TP, Hu B, Walsh CP, Li J, Tang F, Xu GL
    The epigenomes of mammalian sperm and oocytes, characterized by gamete-specific 5-methylcytosine (5mC) patterns, are reprogrammed during early embryogenesis to establish full developmental potential. Previous studies have suggested that the paternal genome is actively demethylated in the zygote while the maternal ge...

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