Table 2:

Generalized protocol table summarizing HTS assays.

Generalized Protocol Table
Step	Parameter	Value	Description
1	Reagent	2 μL	SMMTase Enzyme (2x) in reaction buffer, columns 1–48
2	Reagent	1 μL	SAM (4x) in reaction buffer, columns 1–48
3	Controls	23 nL	DMSO in column 4; sinefungin in DMSO (0 μM – 40 μM) in 7-point 1:2 dilution series (n = 2) in column 2
4	Library compounds	23 nL	Columns 5–48
5	Reagent	1 μL	Substrate (4x) in reaction buffer, columns 2–48
6	Time	20–30 min	Incubation
7	Reagent	1 μL	MTase-Glo reagent (5X), columns 1–48
8	Time	30 min	Incubation
9	Reagent	5 uL	MTase-Glo Detection reagent, columns 1–48
10	Time	30 min	Incubation, luminescence evolution
11	Detection	Luminescence	ViewLux uHTS Microplate Imager (PerkinElmer)
Step	Notes
1, 2, 5	White Medium Binding 1536-well plates (white, flat bottom, medium binding Cat# AWK010000A, Aurora Microplates, Scottsdale, AZ), Reaction buffer: 50 mM Tris, pH 8.0, 3 mM MgCl2, 1 mM EDTA, 50 mM NaCl, 1 mM DTT, and 0.1 mg/mL BSA; refer to Tables 2 and S1 for specific reagent concentrations
3, 4	Pintool transfer
6, 8, 10	Room temperature
6	Final reaction conditions: 10 nM COMT, 5 μM SAM, 15 μM norepinephrine, 50 mM Tris, pH 8.0, 3 mM MgCl2, 1 mM EDTA, 50 mM NaCl, 1 mM DTT, and 0.1 mg/mL BSA; refer to tables 2 and S1 for specific timing
8	Conversion of SAH to ADP; MTase Glo Kit (Promega, Madison, WI)
10	Conversion of ADP to ATP and detection by UltraGlo luciferase; MTase Glo Kit (Promega, Madison, WI)
11	Settings: 20 s exposure, 1X binning, high gain, medium speed