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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
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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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