In vitro interactions between biotinylated nucleosomes and His-GFP tagged DNMT proteins were assessed by luminescence proximity AlphaScreen assay (PerkinElmer) as described40 (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.
Nickel chelated acceptor beads
Manufactured by PerkinElmer
Nickel-chelated acceptor beads are a type of laboratory equipment used in various biochemical and biophysical assays. These beads are coated with nickel ions, which can bind to proteins or other biomolecules containing histidine tags. The primary function of these beads is to serve as a solid-phase platform for capturing and immobilizing target analytes, enabling efficient separation and detection in various assay formats.
Automatically generated - may contain errors
2 protocols using nickel chelated acceptor beads
1
Quantifying Protein-Nucleosome Interactions
2
Quantifying Protein-Nucleosome Interactions
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In vitro interactions between biotinylated nucleosomes and His-GFP tagged DNMT proteins were assessed by luminescence proximity AlphaScreen assay (PerkinElmer) as described40 (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.
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