Anti-5-hydroxymethylcytosine (5hmC) Antibody, clone AB3/63.3

Four types of cytosine modifications have been identified in mammalian DNA, namely 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). While 5mC, 5hmC, and 5fC occur in embryonic stem (ES) cells and many cell types in all the major organs (e.g. brain, heart, lung and kidney), 5caC has only been detected in a few cell types at a 10 times lower level than 5fC. The sequential oxidation of 5mC to 5hmC, 5fC, and 5caC catalyzed by the ten-eleven translocation (TET) enzymes has been proposed as a potential route for the demethylation of 5mC back to cytosine (C). Thymine DNA glycosylase (TDG), for example, can excise 5fC and 5caC from DNA as part of the base excision repair (BER) pathway to restore the C-nucleotide. Emerging evidences suggest that the different modifications may be selectively recognized by various proteins, and methyl-CpG-binding protein 2 (MeCP2) binding to 5hmC has been proposed as a means of epigenetic regulation in neurons. The conversion of 5hmC to 5fC, in turn, induces an enhanced binding of DNA-repair-associated proteins to 5fC, which has been proposed as a signal for DNA demethylation.

Variable, depending on the size(s) of the DNA molecule(s) with 5-hydroxymethylcytosine (5hmC) modification.

5-Hydroxymethylcytosine conjugated to BSA

Anti-5-hydroxymethylcytosine (5hmC) Antibody, clone AB3/63.3 is a rat monoclonal antibody that detects 5hmC modification and has been validated in Dot Blot, Immunocytochemistry, and Chromatin Immunoprecipitation (ChIP).

Chromatin Immunoprecipitation (ChIP) Analysis: A representative lot detected 5-hydroxymethylcytosine (5hmC) enrichment sites using liver chromatin preparations from wild-type female mice and mice with liver-specific Hnf4a knockout. A lower 5hmC modification level at Slc22a6 exon1 and Mgst3 intron1–2 was found associated with Hnf4a deficiency (Zhang, Q., et al. (2014). PLoS One. 9(1):e84925).

Immunocytochemistry Analysis: A representative lot detected embryogenesis stage-dependent 5hmC immunoreactivity by indirect fluorescent staining of 4% paraformaldehyde-fixed, 0.5% Triton X-100-permeabilized, 2N HCl-DNA denatured porcine iPS and advanced pronuclear embryos of different origins (Cao, Z., et al. (2014). Theriogenology. 81(3):496-508).

Research Category
Epigenetics & Nuclear Function

Research Sub Category
Chromatin Biology

Target modification is not species-specific.

This antibody detects 5-hydroxymethylcytosine, but not 5-methylcytosine or unmethylated cytosine.

Format: Purified

Protein G Purified

Purified rat monoclonal IgG2aκ in buffer containing 0.1 M Tris-Glycine (pH 7.4), 150 mM NaCl with 0.05% sodium azide.

Stable for 1 year at 2-8°C from date of receipt.

Control
Unmodified DNA, 5-Methylcytosine DNA, and 5-hydroxymethylcytosine DNA

Evaluated by Dot Blot in a panel of of DNA cytosine modifications.

Dot Blot Analysis: 1-10 µg/mL of this antibody detected 10-50 ng of DNA with 5-hydroxymethylcytosine (5hmC) modification, but not DNA with only unmodified cytosine or 5-methylcytosine (5mC) modifiction.

Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.