M5 multi mode plate reader

Peptides of the first twenty amino acids of H3 with and without trimethylation at lysine position 9 and C-terminal fluorescein modification were synthesised and purified to greater than 95% purity (Francis Crick Institute, Chemical Biology Science Technology Platform). Initial binding assays using 6xHis-MBP-DNMT3B 1–555 protein showed a dose-response increase in fluorescence that was not observed in protein or peptide-alone samples. A full 1.5-fold titration series from 23 nM to 12 μM of 6xHis-MBP-DNMT3B 1–555 was incubated with 20 nM of fluorescent unmodified or methylated peptide in 20 mM HEPEs pH 7.5, 100 mM NaCl, 0.005% NP-40, 10 μM ZnCl₂, 1 mM DTT, 2.5% glycerol). Assays were performed in 25 μl reactions in a 384-well plate covered and incubated for 1 h at 18 °C. Fluorescence was measured with 480 nm excitation and 540 nm polarised filters (cut-off at 530 nm) in an M5 multimode plate reader (Molecular Devices). The resulting values were background subtracted (peptide only) and normalised by dividing relative fluorescent units by the highest concentration response to give fraction bound. Two technical repeats and two biological repeats were plotted in GraphPad Prism with a non-linear regression, and Bmax defined as 1.