Table 3.

Dissociation-Enhanced Lanthanide Fluorescence Immunoassay Protocol Table

Step	Parameter	Value	Description
1	Plate cells	45 μL	5,000 PC3 cells
2	Incubation time	Overnight	37°C, 5% CO2
3	Control	5 μL	DMSO
4	Library compounds	5 μL	DZNep, RBC124
5	Incubation time	3 days	37°C, 5% CO2
6	Remove media
7	Freeze plates	>1 h	−80°C
8	Lysis buffer	50 μL	0.2 N HCl
9	Incubation time	Overnight	4°C
10	Transfer lysate	20 μL
11	Neutralization buffer	5 μL
12	Incubation time	1 h	Ambient temperature
13	Wash plates	50 μL once, 100 μL twice	TBS-T
14	Blocking	100 μL	SuperBlock blocking buffer
15	Incubation time	1 h	Ambient temperature
16	Wash plates	100 μL three times	TBS-T
17	Primary antibody	25 μL	1 μg/mL rabbit anti-H3K27me3 or 0.29 μg/mL rabbit anti-histone H3
18	Wash plates	100 μL three times	TBS-T
19	Secondary antibody	25 μL	europium-N1-labeled anti-rabbit IgG antibody
20	Wash plates	100 μL three times, 120 μL two times	TBS-T
21	Enhancement solution	25 μL
22	Incubation time	30 min	Ambient temperature
23	Assay readout	320 and 615 nm	EnVision plate reader, TRF mode

Step Notes

1. 384-well clear-bottom Greiner Bio-One assay plate.

2,5. Plates were sealed with sterile breathable tape.

4. 100 μM to 15 nM dilution series.

6. Use aspiration.

9,12,15. Plates lidded.

10. Transfer cell lysates to two 384-well high-binding MaxiSorp plates.

17,19. Diluted in SuperBlock-T20.

22. Plates lidded until read.

23. Excitation with laser (320 nm), emission filter (615 nm).

HCl, hydrochloric acid; TBS-T, Tris-Buffered Saline Tween-20; TRF, time-resolved fluorescence technology.