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Anti-5-Hydroxymethylcytosine Polyclonal Antibody

Low cross reactivity with Cytosine and 5-methylcytosine versus other available antibodies.
Can be used with a variety of genomic DNA sources.
High sensitivity to low masses of 5-Hydroxymethylcytosine DNA.

Product Size Catalog # Price Qty
Anti-5-Hydroxymethylcytosine Polyclonal Antibody (25g) 1g /ul 25 µg/25 μl A4001-25
Anti-5-Hydroxymethylcytosine Polyclonal Antibody (50g) 1g /ul 50 μg/50 μl A4001-50
Anti-5-Hydroxymethylcytosine Polyclonal Antibody (200g) 1g /ul 200 μg/200 μl A4001-200

About Anti-5-Hydroxymethylcytosine Polyclonal Antibody

Recent advances in the field of epigenetics have identified 5-hydroxymethylcytosine (5-hmC) as a key factor in the regulation of gene expression, with substantial implications in the study of tissue differentiation, neurological development, and carcinogenesis. Studies of this epigenetic marker are typically confounded by a lack of reliable methodology for differentiation from the highly prevalent 5-methylcytosine in a DNA sample. The rabbit Anti-5-hydroxymethylcytosine polyclonal antibody has been developed in order to robustly distinguish between hydroxymethylated DNA and methylated or unmodified DNA. Specificity of the antibody is enhanced such that crossreactivity with unmodified and methylated templates is suppressed to near-background levels. The antibody has been extensively tested and validated in ELISA and immunoprecipitation-based enrichment assays, and is suitable for use in further applications including immunohistochemical labeling and chromatographic blotting.

Format Purified from serum and dialyzed.
Concentration 1 mg/ml per BCA quantitation.
Source Rabbit
Storage Store at -20 °C with prolonged storage at -80 °C. Avoid repeated freeze/thawing.
Reaction Conditions With proper storage, the antibody is stable for one year from the date of receipt.
Specificity 5-hydroxymethylcytosine in single-stranded DNA from any mammalian, plant, insect, or microbial sources, as well as artificial templates used for standardization.
Buffer Solution Antibody is provided in PBS at pH 7.5.
Isotype IgG1
Product Type Polyclonal Antibody

Hydroxymethylated DNA is efficiently enriched using the anti-5-hydroxymethylcytosine antibody. DNA was immunoprecipitated from 1 ng of a mixed non-methylated/methylated/hydroxymethylated (10:1:1) DNA population. This population was comprised of a mixture of non-methylated plasmid DNA, a methylated version of the same plasmid containing a point mutation that introduces a BamHI restriction site, and a hydroxymethylated version of the same plasmid with a KpnI restriction site. After IP, the region of DNA containing the restriction site was amplified by PCR, digested with either BamHI (B) or KpnI (K), and visualized in a 1.4% (w/v) agarose/TAE/EtBr gel. The results indicate high sensitivity of the antibody for 5hmC DNA with no detectable crossreactivity to 5mC DNA

The anti-5-hydroxymethylxytosine polyclonal antibody demonstrates high sensitivity to 5-hmC in genomic DNA. In a standard ELISA workflow, 100 ng of purified genomic DNA from several murine tissue sources — brain, kidney, liver, and thymus — are coated per well. The anti-5-hydroxymethylxytosine antibody and the secondary antibody (anti-rabbit horseradish peroxidase conjugate) are used at 1:1,000 dilution in modified TBS and incubated at 37°C for 1 hour. Optical density was measured after 1 hour of ABTS substrate incubation at room temperature. Independent quantitation of 5-hmC levels using LC/MS analysis of gDNA from the same tissue sources indicates brain at 0.548 %, kidney at 0.225%, liver at 0.107 %, and thymus at 0.030% (refer to Catalog #D5019), demonstrating high correlation with the colorimetric ELISA data above

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