Table 1.

Steps of the DNA Ligase IV Assay with the Scintillation Proximity Assay Method

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.