Table 1.
AlphaLISA Assay Protocol Table
| Step | Parameter | Value | Description |
|---|---|---|---|
| 1 | Plate cells | 10 μL | 2,500 Su-DHL-6 cells |
| 2 | Control | 5 μL | DMSO |
| 3 | Library compounds | 5 μL | DZNep, RBC124 |
| 4 | Incubation time | 3 days | 37°C, 5% CO2 |
| 5 | Lysis buffer | 5 μL | |
| 6 | Incubation time | 15 min | Ambient temperature |
| 7 | Extraction buffer | 10 μL | |
| 8 | Incubation time | 10 min | Ambient temperature |
| 9 | Acceptor beads+antibody | 10 μL | 100 μg/mL Acceptor beads; 15 nM antibody |
| 10 | Incubation time | 1 h | Ambient temperature |
| 11 | Donor beads | 10 μL | 100 μg/mL Donor beads |
| 12 | Incubation time | 2 h | Ambient temperature |
| 13 | Assay readout | 680 and 570 nm | EnVision plate reader, AlphaScreen mode |
Step Notes
1. 384-well white opaque CulturPlates.
3. 100 μM to 15 nM dilution series.
4. Plates were sealed with sterile breathable tape.
5,7. From the H3K27me3 Cellular Detection Kit.
6,8,10. Plates lidded.
9,11. In 1× Cell-Histone Detection buffer from the H3K27me3 Cellular Detection Kit.
12. Plates lidded until read.
13. Excitation with laser (680 nm), emission filter (570/100 nm).
DMSO, dimethylsulfoxide; DZNep, 3-Deazaneplanocin A.