Specificity of the slot blot assay using MBD1 protein. (a) Methylation status of the λDNA assessed by Hpa II endonuclease restriction analysis shows that in vitro methylated λDNA (M+) was resistant to endonuclease cleavage, but unmethylated λDNA (M-) was not. (b) Methylated λDNA (100%) was added in increasing amounts to unmethylated λDNA (0%) to create a dilution series of methylated DNA from 0% to 100% in a total amount of 500 ng. The blot was stained with methylene blue to ensure the equal loading of total DNA. (c) The same blot detected by MBD1. (d) The observed band intensity was plotted against the methylation percentage. The regression equation with an adjusted R2 of 0.9705 was presented on figure.