Table 1.

TGR Assay Protocol

Step	Parameter	Value	Description
1	Reagent	3 μl	Enzyme and no-enzyme solutions
2	Library compounds	23 nl	57.2 μM–28 nM titration series
3	Incubation time	15 min	Compound interaction with targets
4	Reagent	1 μl	Second equivalent of NADPH
5	Reagent	1 μl	Substrate
6	Assay readout	405 nm	Absorbance kinetic read

Step notes
1	Black clear-bottom plates, 15 nM TGR and 100 μM NADPH final concentrations.
2	Pintool transfer of DMSO solutions of test compounds.
3	Room temperature incubation.
4	Solution of 500 μM NADPH; necessary in order to minimize the effect of noninhibitory redox cyclers that might exhaust NADPH during step 3 and thus give the erroneous appearance of inhibition.
5	DTNB at 3 mM final concentration.
6	ViewLux CCD imager (PerkinElmer), 405/10 nm excitation filter, excitation energy 4,000, exposure time 2 s, clear emission filter, one read per 120 s for 8 min