5-Hydroxymethylcytosine (5-hmC, 5-hydroxymethylcytidine) antibody tested by Methyl DNA immunoprecipitation. DNA (25 pg) derived from the promoter of the APC gene was spiked into 500 ng of human genomic DNA and subjected to the MeDIP procedure using 1 μl of 5-Hydroxymethylcytidine antibody (5hmC, maroon bars) or 1 μl of control rabbit IgG (IgG, blue bars). Real time quantitative PCR was performed on the immunoprecipitated DNA and results plotted as % of input DNA. The spiked APC DNA contained either no methylation (DNA), 5-methylcytosine methylation (5-mC) or 5-hydroxymethylcytosine methylation (5-hmC).
5-Hydroxymethylcytosine (5-hmC, 5-hydroxymethylcytidine) antibody tested by dot blot analysis. DNA samples (10 ng or 50 ng as indicated) were spotted onto positively charged nylon membrane and blotted with 5-Hydroxymethylcytidine antibody at a dilution of 1:10,000. Top Panel: Double stranded DNA. Bottom Panel: Single stranded DNA. Lane 1: Unmethylated DNA. Lane 2: DNA containing 5-methylcytosine. Lane 3: DNA containing 5-hydroxymethylcytosine.
5-Hydroxymethylcytosine (5-hmC, 5-hydroxymethylcytidine) antibody tested by dot blot analysis. DNA samples were spotted onto positively charged nylon membrane and blotted with antibodies as indicated. Panel A: 5-Hydroxymethylcytidine antibody recognizing 5-hydroxymethylcytosine (1:10,000 dilution). Panel B: 5-Methylcytidine antibody (1:1,000 dilution). Lane 1: DNA derived from mouse embryonic stem cells (150 ng). Lane 2: DNA derived from mouse spleen (600 ng). Lane 3: 27 base oligonucleotide containing 5-hydroxymethylcytosine (1.2 ng). Lane 4: 33 base oligonucleotide containing 5-methylcytosine (2000 ng).
