| Assay Method Information | |
| | Determination of CRBN-Binding Affinity of Compounds |
| Description: | 1. According to the instructions of the CEREBLON BINDING kits, the compounds to be tested of the present invention and lenalidomide were serially diluted using diluent #9 (1×) solution to obtain a final concentration of 2 μM for both the tested compounds and lenalidomide solution.2.2.5 μL of the above 2 μM of the tested compounds and lenalidomide solution, as well as the same volume of diluent #9 (1×) solution (solvent control group, Std0) were taken and added to each well of a 96-well plate, respectively. Then, 2.5 μL of (human Cereblon WT GST-tagged protein) solution was added to each well. Finally, 5 μL of the uniformly mixed thalidomide-Red reagent and GST Eu antibody working solution were added to each of the above wells. The final concentration of the tested compounds and lenalidomide in each well is 0.5 μM.3. The blank control wells were sequentially added with 2.5 μL of diluent #9 (1×) solution, 2.5 μL of PROTAC binding buffer, and 5 μL of uniformly mixed thalidomide-Red reagent and GST Eu antibody working solution.4. After sealing and incubating the solutions in the above wells at room temperature for 3 hours, the absorbance values at emission wavelengths of 620 nm and 665 nm were detected by the HTRF method using a Spark microplate reader (V3.1 SP1). |
| Affinity data for this assay | |
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