| Assay Method Information | |
| | TR-FRET BRD4 Binding Assay |
| Description: | The BRD4 bromodomain 1 and/or 2 binding activity were measured by using the Cayman BRD4 bromodomain 1 or 2 TR-FRET Assay Kit (Item. No. 600520, 600530, Ml, USA) respectively, according to the manufacturer's instructions. Briefly, compounds were serial diluted into the 1×TR-FRET Assay Buffer with no more than 8% DMSO, and the final concentration tested was distributed from 1 M to 10 uM with no more than 2% DMSO. (+)JQ-1 was used as positive control. Pre-incubate the control or compounds with the bromodomain (1 or 2) Europium Chelate in the 384-well plate for 15 min at room temperature (Protect from light). Then the reconstituted BRD4 bromodomain ligand/APC Acceptor Mixture were added to every well and incubate for 1 hr at room temperature. The plate was sealed with adhesive aluminum seal to prevent photobleaching. Then the plate was read in time-resolved format by exiting the well at 340 nm and reading emmission at 620 and 670 nm, using 100 us delay and 500 us read window. Data analysis was performed using the TR-FRET ratio which is the 670 nm emission/620 nm emission. |
| Affinity data for this assay | |
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