Table 1.
Step | Parameter | Value | Description |
---|---|---|---|
1 | Library compounds | 1 μL | 100 μM in 10% DMSO (v/v) |
2 | Control compounds | 1 μL | 10% DMSO (v/v) |
3 | Enzyme | 5 μL | 1 nmol His-Lig4-[α-33P]-AMP |
4 | Incubation time | 15 min | Room temperature |
5 | Substrate | 6 μL | 250 pmol dsDNA substrate |
6 | High control | 6 μL | 250 pmol dsDNA substrate |
7 | Low control | 6 μL | Reaction buffer (60 mM Tris-HCl [pH 8.0] and 10 mM MgCl2) |
8 | Incubation time | 15 min | Room temperature |
9 | Imaging beads | 50 μL | HIS TAG PS Imaging beads |
10 | Incubation time | 24 h | 4°C—cold temperature |
11 | Centrifugation | 1 min | 2,000 rpm |
12 | Assay readout | Epi mirror: empty Emission mirror: empty |
LEADseeker multimodality imaging system |
Step Notes
1–2. Dispensing on the PP-384-M Personal Pipettor using a custom 384 head
3. Dispensing with the FlexDrop IV
4. Plates incubated at room temperature for 15 min
5 to 7. Dispensing with the FlexDrop IV
8. Plates incubated at room temperature for 15 min
9. Dispensing with the Multidrop 384
10. Plates incubated at cold temperature for 24 h
11. Plates centrifuged at 2,000 rpm for 1 min
12. Radiometric signal quantified with empty epi mirror and empty emission mirror with [14C]-plate as a reference
DMSO, dimethyl sulfoxide; dsDNA, double-stranded DNA.