ELISA of single stranded DNA using rabbit monoclonal anti-5-mC (clone RM231) antibody. 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 RM231 was used as the primary antibody, and an alkaline phosphatase conjugated anti-rabbit IgG as the secondary antibody.
Dot blot of double stranded DNA using rabbit monoclonal anti-5-mC (clone RM231) antibody. 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 RM231.
Immunocytochemical staining of HeLa cells using rabbit monoclonal anti-5-mC (clone RM231) antibody (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-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 RM231 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.
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