| Assay Method Information | |
| | BET BRD Assay |
| Description: | 3x Complete Substrate plus Inhibitor Solution in Assay Medium (Opti-MEM I Reduced Serum Medium, no phenol red, and no serum) was prepared just before measuring BRET. This solution consisted of a 1:166 dilution of NanoBRET Nano-Glo Substrate plus a 1:500 dilution of Extracellular NanoLuc Inhibitor in Assay Medium. For a 96-well plate, 30 ul of NanoBRET Nano-Glo Substrate, 10 ul of Extracellular NanoLuc Inhibitor and 4,960 ul of Assay Medium were mixed to produce 5 ml of 3x Complete Substrate plus Inhibitor Solution, followed with gently mixing by inversion 5-10 times in a conical tube. (The final concentration of Extracellular NanoLuc Inhibitor in the 3x solution was 60 uM, for a working concentration of 20 uM. Use 3x Complete Substrate plus Inhibitor Solution within 2 hours. Discard any remaining solution). 50 ul of 3x Complete Substrate plus Inhibitor Solution were added to each well of the 96-well plate, followed by incubation for 2-3 minutes at room temperature. Donor emission wavelength (e.g., 450 nm) and acceptor emission wavelength (e.g., 610 nm) were measured by using the GloMax Discover System or other NanoBRET Assay-compatible luminometer (it is recommended measuring BRET within 10 minutes after adding NanoBRET Nano-Glo Substrate plus Extracellular NanoLuc Inhibitor Solution. However, BRET can be measured for up to 2 hours, but there will be some loss of luminescence signal). To generate raw BRET ratio values, the acceptor emission value (e.g., 610 nm) was divided by the donor emission value (e.g., 450 nm) for each sample [to correct for background, the BRET ratio was subtracted in the absence of tracer (average of no-tracer control samples) from the BRET ratio of each sample]. |
| Affinity data for this assay | |
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