Anti–5–caC polyclonal antibody

Catalog #

25201

$415 *

Size: 100 µg

Qty

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Description

Polyclonal antibody raised in rabbit against 5–Carboxylcytosine (5ca–CMP monophosphate) conjugated to BSA

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Synonyms

5–Carboxylcytosine

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Product Data Gallery

Product Info

Storage and Usage

Citations

Clonality

polyclonal

Host Species/Expression System

Rabbit

Purification

Protein A affinity purified

Format

Aqueous buffer solution

Formulation

PBS (pH 7.4), 30% glycerol, 0.05% sodium azide

Concentration

20 µg

Immunogen

nucleotide

Background

The so-called 6th base, 5–hydroxymethylcytosine has been discovered in 2009. The function of this new modified base remains mostly unclear, but it is certain that the 5–hmC 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.

Storage/Stability

Store at –80°C for up to 2 years. Centrifuge after first thaw to maximize product recovery. Aliquot to avoid repeated freeze/thaw cycles. Aliquots may be stored at –20°C for at least one month.

Assay Conditions

Dot blot analysis using the antibody directed against 5-caC
To demonstrate the specificity of the antibody against 5-caC (Cat. # 25201), a Dot Blot analysis was performed using synthetic oligonucleotides containing different modified C-bases (indicated in red). 125 and 25 ng of the respective oligos 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 assay using the 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 antibody against 5-caC (Cat. # 25201), diluted 1:500, and with an anti-FLAG antibody, followed by DAPI counterstaining.

Immunoprecipitation using the antibody directed against 5-caC
Immunoprecipitation was performed with the antibody against 5-caC (Cat. # 25201) 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 immunoprecipitated 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.

Applications

DB (1:500 – 1:1000)
IF (1:500)
DNA IP (4 µg genomic DNA/IP)

Shipping Temperature

-20°C or-80°C

Species Reactivity

Cross Reactivity

Specific for 5–carboxylcytosine (5–caC). Does not react with 5–mc, 5–hmc, unmodified cytosine, or 5–formylcytosine (5–fC) and 5–carboxylcytosine (5–caC)

Notes

The optimal antibody concentration should be determined by the end–user.

Warnings

Avoid freeze/thaw cycles

Scientific Category

Antibodies

Regulatory Status

Research Use Only

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Anti–5–caC polyclonal antibody

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