Quantifying Protein-Nucleosome Interactions

In vitro interactions between biotinylated nucleosomes and His-GFP tagged DNMT proteins were assessed by luminescence proximity AlphaScreen assay (PerkinElmer) as described^{40 (link)}. Briefly, 100 nM biotinylated nucleosomes and 100 nM His-tagged proteins were incubated with 5 μg/mL streptavidin-coated donor beads and 5 μg/mL nickel-chelated acceptor beads (PerkinElmer) in 100 μL total volume of AlphaScreen buffer (50 mM MOPS, pH7.4, 50 mM NaF, 50 mM CHAPS, and 0.1 mg/ml BSA) for 1.5 h in the dark at room temperature. Photon counts were determined in 384 plates using an Envision-Alpha Reader (PerkinElmer) (Extended Data Fig. 2a). Each experiment was repeated at least four times. Data were validated for normality by Shapiro-Wilk and Kolmogorov-Smirnov tests and analyzed by one-way ANOVA using GraphPad Prism.