Immunocytochemical analysis of HeLa cells using two different fixation/denaturation conditions and ab214727 at 2 μg/mL dilution (red); actin filaments have been labeled with fluorescein phalloidin (green), and nuclei stained with DAPI (blue).

Chromatin denaturation is required to expose the epitopes in DNA and allow the antibody to efficiently detect 5mC. Stronger denaturing conditions such as HCl (bottom panels) will result in enhanced nuclear staining compared to weaker denaturing conditions such as acetic acid (HAc, top panels).

However, stronger denaturants such as using HCl may alter or degrade other molecules and intracellular structures, which can be problematic for experiments involving multi-color staining or looking at subcellular morphology. For those experiments we would suggest using weaker denaturants such as HAc.

Direct ELISA of HeLa cell genomic DNA using anti-5-mC antibody (RM231). The plate was directly coated with different concentrations of genomic DNA isolated from HeLa cells. 1 ug/mL or 3 ug/mL of ab214727 was used as the primary antibody, and a HRP conjugated anti-rabbit IgG as the secondary antibody.

MeDIP was performed using anti-5-mC antibody (RM231) at a 2:1 DNA:Ab ratio. 1 ng of unmethylated, 5-Methylcytosine (5-mC) or 5-Hydroxymethylcytosine (5-hmC) DNA standard (897 bp) was spiked in 1ug of genomic DNA isolated from HeLa cells as the control. Realtime PCR was then performed to determine the capture of DNA standard as in % of input.

Dot blot of double stranded DNA using ab214727 at 0.5 ug/mL. The membrane was pre-spotted with 50, 5, and 0.5 ng/dot of double stranded 5-Hydroxymethylcytosine (5-hmC) DNA, 5-Methylcytosine  (5-mC) DNA, and unmethylated DNA. The pre-spotted membrane was then blotted with ab214727.

ELISA of single stranded DNA using ab214727 in a serial dilution. The plate was coated with streptavidin and then biotinylated single stranded unmethylated DNA, 5-Methylcytosine (5-mC) DNA, and 5-Hydroxymethylcytosine (5-hmC) DNA. Secondary antibody: alkaline phosphatase conjugated anti-rabbit IgG.


ab214727 staining 5-methylcytosine (5-mC) in human brain tissue sections by Immunohistochemistry (paraformaldehyde-fixed, paraffin-embedded sections). Tissue was blocked with 5% normal goat serum for 60 minutes at 22°C. Samples were incubated with primary antibody (20ng/ml) for 1 hour at 22°C. A HRP-conjugated Goat anti-rabbit IgG polyclonal was used as the secondary antibody.

Flow Cytometry analysis of 5-mC expression in HEK293 cells using ab214727. The cells were fixed with ice-cold MeOH, permeabilized with 0.5% Triton X-100, denatured with 2N HCl, then stained with ab214727 (Blue) or with a negative control antibody (Red).