| Assay Method Information | |
| | SMARCA2 Bromodomain Binary and Ternary Binding Assay |
| Description: | The inhibitory activity of compounds was evaluated in vitro using TR-FRET assay with white 384-well low volume microplate (PerkinElmer ProxiPlate Plus), in which the compound competes the same binding site with the ligand, and thus lead to dose-dependent TR-FRET signal reduction. The cooperativity of the compounds for ternary complex formation with E3 ligase was evaluated in the absence or presence of saturating concentrations of VCB. Testing compounds were dissolved in DMSO at 10 μM and tested in 9-dose IC50. The assay mixture was prepared by mixing SMARCA2 (10 nM final), biotinylated probe (25 nM final), and assay buffer or VCB (5 μM) in 1×AlphaLISA Epigenetics Buffer (PerkinElmer AL008F) with 1 mM TCEP. The compounds in DMSO were added to each well in 3-fold serial dilution by dispenser (TECAN D300E) and incubate for 20 minutes at room temperature before addition of detection reagents, Lance Eu W1024 anti-6×His (0.6 nM final, PerkinElmer AD0110) and Streptavidin Surelight APC (6 nM final, PerkinElmer CR130-100). The plate was then sealed and further incubated at 4° C. overnight in dark, and then was read by Envision multimode plate reader (PerkinElmer, 2102-0010). The ratio of florescence signal at 665/620 was used in data analysis. Percentage inhibition was calculated by % inhibition=100×(FDMSO-F)/(FDMSO-FPC), in which FDMSO is DMSO control, and FPC is positive control. IC50 values were determined from dose response curve by fitting the percent inhibition against compound concentration using |
| Affinity data for this assay | |
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