| Assay Method Information | |
| | CRBN TR-FRET Assay |
| Description: | Table 26: Test compounds dissolved in DMSO are dispensed onto assay plates (Proxiplate 384 PLUS, white PerkinElmer/revvity; 6008289) using an Access Labcyte/Beckman Coulter Workstation with the Labcyte/Beckman Coulter Echo 5- or 6-series. For the chosen highest assay compound concentration of 100uM, 152.5 uL of compound solution is transferred from a 10 mM DMSO compound stock solution. A series of eleven fivefold dilutions per compound is transferred to the assay plate, compound dilutions are tested in duplicates. DMSO is added as backfill to a total volume of 152.5 nL. The assay is run on a fully automated robotic system. Subsequently, 45 uL of the Cy5-labeled CRBN-binder (10 uM stock in 100% DMSO) is added to rows 1-23 and 45 nL of 100% DMSO is added to row 24. To start the assay 15 uL of CRBN reaction mix in assay buffer (20 mM HEPES pH 7.3, Thermo Fisher J16924, 150 mM NaCl, Lonza AccuGene 51202 and 0.005% Tween 20, BioRad 161-0781) including His-Avi-tagged CRBN-DDB1 complex ectopically using a retrovival system. |
| Affinity data for this assay | |
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