Table 1.
Final 1536-well assay protocol
| Step | Parameter | Value | Description |
|---|---|---|---|
| 1 | Reagent | 4 μL | 10 μM ATP buffer |
| 2 | Library compounds | 23 nL | 40 μM to 0.24 nM dilution series |
| 3 | Controls | 23 nL | ATP, compound 1, DMSO |
| 4 | Reagent | 2 μL | Detection Buffer |
| 5 | Incubation time | 8 min | RT incubation |
| 6 | Assay readout | 5 sec | Luminescence read |
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| Step | Notes | ||
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| 1 | Medium-binding white solid Kalypsys plates. 100 μL pre-dispense, four tip dispense columns 1–48. Buffer: 50 mM KCl, 7 mM MgCl2, 10 μM ATP, 0.01% Tween, 0.05% BSA. Mixture kept on ice. | ||
| 2 | Pin-tool transfer compound library for a (final) range of 40 μM to 0.24 nM | ||
| 3 | Pin-tool transfer, Column 1 sixteen-point titrations in duplicate of ATP beginning at 10 μM (final), Column 2, sixteen-point titrations in duplicate of compound 1 beginning at 40μM (final), Column 4, compound 1 at 40 μM (final). Pin-tool transfer tip wash sequence: DMSO,iPA, MeOH, 3-s vacuum dry. | ||
| 4 | Kinase-Glo luminescent reagent containing Ultra-Glo luciferase and high 4 concentrations (~mM) of luciferin. 100 μL pre-dispense, four tip dispense columns 1–48 | ||
| 5 | RT incubation in auxilliary plate hotel | ||
| 6 | PE ViewLux, clear filter | ||