Table 4.

PP1α Assay Protocol

Step	Parameter	Value	Description
1	Enzyme mix	100 μL	PP1α in 1.5× assay buffer
2	Test compounds, control inhibitor, or vehicle control	1.5 μL	Dissolved in DMSO at 100× final concentration
3	Substrate mix	48.5 μL	309 μM OMFP in water
4	Incubation time	30 min	Room temperature
5	Stop solution	50 μL	1 M potassium phosphate (pH 10)
6	Assay readout	Ex: 485 nm, Em: 525 nm	Fluorescence of OMF product

Step Notes

1. Dispensed with stepper pipettor and positive-displacement tip.

2. Dispensed with single-channel pipettor. About 1.5 μL of 10 mM cantharidin was added to low-signal control wells. About 1.5 μL of DMSO was added to high-signal control wells.

3. Dispensed with multichannel pipettor.

4. Final assay mix in 150 μL total volume (30 mM HEPES [pH 7], 0.01% Triton X-100, 0.3 mg/mL BSA, 1 mM sodium ascorbate, 1 mM MnCl₂, 1 mM DTT, 50 μM OMFP, and 3 nM PP1α).

5. Dispensed with multichannel pipettor.

6. SpectraMax^® M5: 515-nm emission filter.