| Assay Method Information | |
| | Biological Activity Primary Activity Assay |
| Description: | NanoBRET assay was carried out according to the manufacturer's suggested protocol (Promega, Madison, WI). HEK293 cells were transfected using NanoLuc-BRD4-BD1 or NanoLuc-BRD4-BD2 fusion vectors and incubated at 37° C. in an atmosphere of 5% CO2 for 20-24 hours. The transfected cells were then dispensed into 96-well plates using 90 μl cell suspension per well containing 2×105 cells/mL in Opti-MEM and 1× final concentration of tracer. 90 μL per well of cell suspension without tracer was also dispensed into at least 3 wells as “No tracer control samples” for background correction. Serially diluted test compounds were prepared at 1000× concentration in DMSO and further diluted to 10× concentration in Opti-MEM. 10 μL per well of the serially diluted 10× test compound was added to the 96-well plates containing cells with 1× tracer, to give a final top concentration of test compound of 10 μM. Plates were then incubated at 37° C. +5% 002 incubator for 2 hours. Immediately prior to BRET measurements, a 3× solution consisting of 1:166 dilution of Nano-Glo® Substrate plus a 1:500 dilution of Extracellular NanoLuc Inhibitor in Opti-MEM was prepared and 50 μL per well was added to the cells. Donor emission (450 nm) and acceptor emission (610 nm) were measured using PHERAstar (BMG LabTech). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |