(A) Enzyme concentration. The assays were carried out for 16 h with the varied concentrations of (▲) SET7/9, (●) SET8, (■) SETD2 and (◆) EuHTMase1.The final concentrations of 150 nM for SET7/9, 1.5 μM for SET8, 250 nM for SETD2, and 10 nM for EuHMTase1 were chosen for HTS; (B) Reaction time. Using these final concentrations, the maximal conversions were reached in 3 h for SET7/9 (▲), 8 h for SET8 (●), 4 h for SETD2 (■), and 1 h for 10 nM EuHMTase1 (◆); (C) DMSO tolerance. The reactions were carried out with 150 nM SET7/9 for 3 h, 1.5 μM SET8 for 8h, 250 nM SETD2 for 4h, and 10 nM EuHMTase1 for 1 h in the presence of a varied amount of DMSO: without (white), 1% (gray), 2% (horizontal lines), 3% (diagonal lines), 5% (black). The four PMTs were tolerant up to 5% DMSO; (D) Enzyme stability. Experiments were performed with 150 nM SET7/9, 1.5 μM SET8, 250 nM SETD2, and 10 nM EuHMTase1. All enzymes were pre-incubated at ambient temperature for 0 (black), 6 (gray), and 16 h (diagonal lines) prior to the assay start. The unchanged reactivity indicated that the four PMTs are stably functional under our assay conditions for at least 16 h; (E) SPA readout under high and low controls. The reactions were carried out under the optimized assay conditions with either active PMTs (black) or HClO4-treated PMTs (white). Upon mixing with 0.2 mg SPA beads, the scintillation signals were recorded with LEADseeker™ Multimodality Imaging System. The ratios of the high-to-low controls of 8 for SET7/9, 3 for SET8, 6 for SETD2, and 30 for EuHTMase1, display an excellent signal-to-noise separation for the present assay format (10 repeats for each data set). Each assay point was performed in triplicate (n=3), and SDs were plotted unless otherwise mentioned.