ELISA of single stranded DNA using Anti-5-hydroxymethylcytosine Antibody [RM236]. The plate was coated with streptavidin and then biotinylated single stranded unmethylated DNA, 5-Methylcytosine (5-mC) DNA, and 5-Hydroxymethylcytosine (5-hmC) DNA. A serial dilution of Anti-5-hydroxymethylcytosine Antibody [RM236] was used as the primary antibody, and an alkaline phosphatase conjugated anti-rabbit IgG as the secondary antibody.
Figure 2: Dot Blot - Anti-5-hydroxymethylcytosine Antibody [RM236] (A121325)
Dot blot of double stranded DNA using Anti-5-hydroxymethylcytosine Antibody [RM236]. 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 un-methylated DNA. The pre-spotted membrane was then blotted with Anti-5-hydroxymethylcytosine Antibody [RM236].
Immunocytochemical staining of HeLa cells using 0.5µg/ml Anti-5-hydroxymethylcytosine Antibody [RM236] (red). Actin filaments have been labeled with fluorescein phalloidin (green). HeLa cells were fixed with 4% paraformaldehyde and permeabilized with methanol (-20°C) before treatment with 2 N HCl for 30 min at 37°C to denature DNA.
Direct ELISA of mouse brain genomic DNA using Anti-5-hydroxymethylcytosine Antibody [RM236]. The plate was directly coated with different concentrations of genomic DNA isolated from mouse brain tissue. 1µg/ml or 3µg/ml of Anti-5-hydroxymethylcytosine Antibody [RM236] was used as the primary antibody, and a HRP conjugated anti-rabbit IgG as the secondary antibody.
hMeDIP was performed using Anti-5-hydroxymethylcytosine Antibody [RM236] at a 10:1 DNA:Antibody ratio. 1ng of unmethylated, 5-Methylcytosine (5-mC) or 5-Hydroxymethylcytosine (5-hmC) DNA standard (897 bp) was spiked in 1µg 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.
Flow cytometry analysis of 5-hmC expression in HEK293 cells using Anti-5-hydroxymethylcytosine Antibody [RM236]. The cells were fixed with ice-cold MeOH, permeabilized with 0.5% Triton X-100, denatured with 2N HCl, then stained with Anti-5-hydroxymethylcytosine Antibody [RM236] (Blue) or with a negative control antibody (Red).