| Assay Method Information | |
| | HTRF (Homogeneous Time-Resolved Fluorescence) Assay |
| Description: | Biochemical potency of compound was determined by using CRBN& DDB1 protein (His Tag). Compounds were tested for blocking the binding of CRBN&DDB1 protein (CRBN, aa 40-442, DDB1, 1-1140, Viva Biotech) with biotin labeled thalidomide in an assay based on the time-resolved fluorescence-resonance energy transfer (TR-FRET) methodology. The assay was carried out in 384-well low volume black plates in a reaction mixture containing CRBN& DDB1 protein, 30 nM biotin labeled thalidomide and 0-10 μM compound in buffer containing 50 mM HEPES pH7.5, 50 mM NaCl, 0.01% BSA, 1 mM DTT and 0.015% Brij-35. The protein was preincubated with compound for 60 minutes at room temperature and biotin labeled thalidomide was added to plate. After further incubation at room temperature for 60 minutes detection reagents Mab Anti-6His Eu cryptate Gold (Cisbio, Cat #61HI2KLB) and Streptavidin-XL665 (Cisbio, Cat #610SAXLG) were added to plate. Plates were sealed and incubated at room temperature for 1 hour, and the TR-FRET signals (ex337 nm, em665 nm/620 nm) were recorded on a PHERAstar FSX plate reader (BMG Labtech). The inhibition percentage of CRBN& DDB1 protein interaction with biotin labeled thalidomide in presence of increasing concentrations of compounds was calculated based on the ratio of fluorescence at 665 nm to that at 620 nm. IC50 was derived from fitting the dose-response % inhibition data to the four-parameter logistic model by Dotmatics. |
| Affinity data for this assay | |
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