| Assay Method Information | |
| | Binding Affinity Assay |
| Description: | The binding affinity of the described test compounds was determined using RED-tris-NTA His-tag labeled Cereblon ligand binding domain. The labeling of the Cereblon ligand binding domain was performed according to manufacturer's protocol in assay buffer (50 nM protein: 10 nM dye, 5:1 protein to dye ratio). 200 μM compound in assay buffer (10 mM HEPES pH 7.4, 300 mM NaCl, 0.1% Pluronic-147, 10% Glycerol, 5 mM DTT) was created from 10 mM compound stock in DMSO. A 16-point serial dilution was achieved by taking 10 μL of the 200 μM first well and diluting into 10 μL of assay buffer in the second well, which was diluted in 10 μL assay buffer in the third well, and so on. DMSO content of each well matched to 2%. 10 μL of 50 nM CRBN was added to each well and mixed thoroughly by pipetting. Final assay concentration of 25 nM labeled protein and highest compound concentration of 100 μM, 1% DMSO. Samples were incubated at room temperature in the dark for five minutes, then collected in standard Monolith capillaries. Assay was performed on the Monolith NT.155Pico using automated excitation level and medium power on M.O. Control software. |
| Affinity data for this assay | |
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