ELISA of single stranded DNA using Anti-5-hydroxymethylcytosine Antibody [RM231]. 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 [RM231] 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 [RM231] (A121326)
Dot blot of double stranded DNA using Anti-5-hydroxymethylcytosine Antibody [RM231]. 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 [RM231].
Immunocytochemical staining of HeLa cells using Anti-5-hydroxymethylcytosine Antibody [RM231] (red). Actin filaments have been labeled with fluorescein phalloidin (green), and nuclei stained with DAPI (blue).
Direct ELISA of HeLa cell genomic DNA using Anti-5-hydroxymethylcytosine Antibody [RM231]. The plate was directly coated with different concentrations of genomic DNA isolated from HeLa cells. 1µg/ml or 3µg/ml of Anti-5-hydroxymethylcytosine Antibody [RM231] was used as the primary antibody, and a HRP conjugated anti-rabbit IgG as the secondary antibody.
MeDIP was performed using Anti-5-hydroxymethylcytosine Antibody [RM231] at a 2:1 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. Real-time PCR was then performed to determine the capture of DNA standard as in % of input.
Flow cytometry analysis of 5-mC expression in HEK293 cells using Anti-5-hydroxymethylcytosine Antibody [RM231]. 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 [RM231] (Blue) or with a negative control antibody (Red).