| 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 pH7.4, 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 ng/ml. Plates were incubated 1 hour and read on a PerkinElmerTM Envision plate reader (680 nm excitation and 570 nM emission settings). |
| Affinity data for this assay | |
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