| Assay Method Information | |
| | Cereblon Binding Assay |
| Description: | Competition Assay: Test compounds in 100% dimethyl sulfoxide were plated onto white, low volume 384 well plates (PerkinElmer Proxiplate 384 plus #6008289) using an Labcyte Echo acoustic dispenser, in an 11-point dose curve and 3-fold dilution scheme. This resulted in 10 μM top dose (starting concentration) and 0.1% DMSO final concentrations after the addition of 10 μL of an assay mixture containing His-tagged Cereblon/DDB1 complex and 50 μM biotin-labeled ligand in a buffer (buffer: 10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20 and 10 mM DTT). Plates were incubated for 30 minutes at room temperature. Detection: 10 μL detection reagents were added in low light conditions containing nickel chelate acceptor beads and streptavidin donor beads (Perkin Elmer 6760002), resulting in a final concentration 20 μg/ml. Plates were incubated 1 hour and read on a PerkinElmer™ Envision™ plate reader (680 nm excitation and 570 nM emission settings). The IC50 of each compound was determined by calculating percent inhibition from no drug and no protein controls, and the Ki of each compound was calculated using the Cheng-Prusoff transformation. |
| Affinity data for this assay | |
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