hMeDIP results
hMeDIP was performed using the hMeDIP kit (mouse monoclonal antibody). The IgG isotype antibody from mouse was used as negative control. 1 µg of Hela cells DNA was prepared and sonicated with Bioruptor® to obtain DNA fragments of 300-500 bp. Unmethylated, methylated and hydroxymethylated spike-in controls have been used. Finally qPCR using specific primer pairs for the unmethylated, methylated and hydroxymethylated DNA sequences has been performed.
Immunofluorescence results obtained with 5-hmC monoclonal antibody (rat)
Mice NIH-3T3 cells have been grown on coverslips. Cold methanol fixation 10 min. HCl treatment 30 min at 37°C. Neutralisation by borate buffer. Blocking 30 min in PBS-BSA Incubation with 5-hmC monoclonal antibody (rat), dilution 1/1000. Overnight incubation at 4°C. Washes with PBS-BSA. Incubation with secondary anti-rat antibody FITC (1/1500). 45 minutes incubation at room temperature. Washes with PBS. DAPI staining and washing with PBS. Mounting in vectashield.
Dotblot analysis of the Diagenode 5-hmC rabbit polyclonal antibody with the C, mC and hmC PCR controls
100 to 4 ng (equivalent of 5 to 0.2 pmol of C-bases) of the hmC, mC and C PCR controls from the Diagenode "5-hmC, 5-mC & cytosine DNA Standard Pack" (Cat No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with the rabbit 5-hydroxymethylcytosine polyclonal antibody (dilution 1:200). The membranes were exposed for 30 seconds.
Dotblot analysis of the Diagenode 5-hmC mouse monoclonal antibody with the C, mC and hmC PCR controls
200 to 2 ng (equivalent of 10 to 0.1 pmol of C-bases) of the hmC (1), mC (2) and C (3) PCR controls from the Diagenode "5-hmC, 5-mC & cytosine DNA Standard Pack" (Cat No. AF-101-0002) were spotted on a membrane (Amersham Hybond-N+). The membrane was incubated with 2 µg/ml of the mouse 5-hydroxymethylcytosine monoclonal antibody (dilution 1:500). The membranes were exposed for 30 seconds.
