HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) cells 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)stained for 5-carboxylcytosine (5-caC) using ab231801 at a dilution of 1/500 in ICC/IF.

Immunoprecipitation was performed with ab231801 on 2 μg of J1 ES genomic DNA, spiked with 1 pg of a control DNA fragment (approximately 700 bp from the RFP (Ring fnger protein) gene) containing different cytosine modifcations.

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 purifed Tet2. The IP’d DNA was subsequently anaysed by qPCR using primers specifc for the control DNA fragments and for GAPDH, used as a negative control. Image shows the enrichment calculated as the ratio of the recovery of the control DNA versus the recovery of the GAPDH negative control.

To demonstrate the specifcity of ab231801 a Dot Blot analysis was performed using synthetic oligonucleotides containing different modifed C-bases (indicated in red).

125 and 25 ng of the respective oligo’s were bound to a Streptavindin-coated multi-well plate. ab231801 was used at a dilution of 1:1,000. The binding of antibody to the DNA was measured by ECL chemiluminescence.