Table 1. .

Assay Protocols for 1536-Well Microtiter Plates Used for qHTS

Step	Kinase-Glo			ADP-Glo
	Parameter	Value	Description	Parameter	Value	Description
1	Reagent	2 µL	ATP/peptide	Reagent	2 µL	ATP/peptide
2	Library	23 nL	0.5 nM–46 µM	Library	23 nL	0.6 nM–55.2 µM
3	Controls	23 nL	TG003	Controls	23 nL	TG003
4	Reagent	1 µL	Clk4	Reagent	0.5 µL	Clk4
5	Time	4.5 h	r.t. incubation	Time	1 h	r.t. incubation
6	Reagent	3 µL	Kinase-Glo	Reagent	2.5 µL	Deplete ATP
7	Read	2 s	ViewLux	Time	45 min	r.t. incubation
8				Reagent	5 µL	ADP ◊ ATP/Luc
9				Time	30 min	r.t. incubation
10				Read	2 s	ViewLux

Step	Notes	Notes
1	100 µM RS peptide 1 µM ATP (final) concentration in buffer: FRD dispense	100 µM RS peptide 1 µM ATP (final) concentration in buffer: FRD dispense
2	Pin-tool transfer compound library for a (final) range of 46 µM–0.5 nM	Pin-tool transfer compound library for a (final) range of 55.2 µM–0.6 nM
3	Pin-tool transfer of controls	Pin-tool transfer of controls
4	Clk4 at 25 nM final, FRD dispense	Clk4 at 25 nM final, FRD dispense
5	Kinase reaction, room-temperature incubation	Kinase reaction, room-temperature incubation
6	Detection Kinase-Glo	ADP-Glo Reagent I
7	Perkin Elmer Viewlux, clear filter	ATP depletion, room-temperature incubation
8		ADP-Glo Reagent II
9		Room-temperature incubation
10		Perkin Elmer Viewlux, clear filter