| Assay Method Information | |
| | Bioluminescence Resonance Energy Transfer (BRET) |
| Description: | Cereblon target engagement was monitored by Bioluminescence Resonance Energy Transfer (BRET) in transfected HEK-293T cells using the NanoBRET TE Intracellular E3 Ligase Assay (Promega). Briefly, 384-well plates (white opaque plates, Corning 3574, low binding surface) were seeded with transfected HEK-293T cells (38 ul/well). 2 uL of 10 uM CRBN tracer (diluted 1:5 in Tracer Dilution Buffer) was added to each well. Plates were centrifuged at 320 g for 1 min at room temperature. Test compounds were added in a 11-point dilution series (typically 10 uM to 100 uM) using a TECAN D300e Digital Dispenser. Plates were shaken for 2 minutes on a microplate shaker to mix compounds. Plates were centrifuged at 320 g for 1 min at room temperature, and subsequently incubated for 2 hours at 37 C.After incubation, plates were allowed to cool to room temperature for 15 minutes. 20 uL of 3X Complete NanoBRET Nano-Glo Substrate plus Inhibitor Solution (Promega, 1:166 Substrate and 1:500 dilution of Extracellular NanoLuc Inhibitor diluted in Opti-MEM) were added to each well, Plates were incubated with shaking at room temperature for 3 minutes covered with foil. Plates were read on a CLARIOstar microplate reader (BMG LabTech), measuring at 450 nm (donor emission) and 610 nm (acceptor emission). |
| Affinity data for this assay | |
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