Quantifying 6mA DNA Methylation via ELISA and Dot-blot

6mA in genomic DNA was measured by ELISA and dot-blot assay. ELISA was performed as in our previous report [17 (link), 18 (link)]. The MethylFlash 6mA DNA Methylation ELISA Kit (Colorimetric) (EpiGentek, NY) was used according to the manufacturer’s instructions. Each sample was run in triplicate. For dot-blot assays, denatured genomic DNA (2 µl, 100 ng) was spotted onto a nylon membrane, UV-crosslinked (20 s, 1200 J/cm²), and blocked in 5% milk for 1 h at room temperature (RT). Then, the membranes were incubated with the 6mA antibody for 4 h, and the secondary antibody for another 1 h at RT. After washing, and the dots was developed and visualized using an ECL detection system (Invitrogen) according to the manufacturer’s protocol. The staining of membranes with methylene blue confirmed the equal DNA loading.