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| | Amplified Luminescent Proximity Homogeneous Assay (ALPHA) |
| Description: | The interactions between test compounds and BRD4 protein containing both bromodomain 1 and bromodomain 2 were measured with human BRD4 protein with N-terminal His tag (BPS Bioscience, San Diego, Calif.) using AlphaScreen® assay at room temperature. A 9 μl reaction mixture in BRD Assay Buffer (BPS Bioscience) containing 25 nM BRD4 and test compounds at various concentrations were incubated for 30 minutes followed by additional 30-minute incubation with 1 μl of 20 M histone H4 peptide (residue 1-21) in the presence of 5% DMSO. Test compounds (see Table 4) were assayed at 10 μM or 31.6 μM for screening purpose, while 8 different concentrations (10 nM-10 μM) were used for IC50 measurements. After the incubation, 20 μl of BRD Detection Buffer (BPS Bioscience) containing 10 μg/ml Glutathione Acceptor beads and 10 μg/ml Streptavidin Donor beads (PerkinElmer, Waltham, Mass.) was added and the mixture was incubated for 50 minutes in darkroom. Binding measurements were taken in duplicate at each concentration using EnSpire® Alpha Multimode Plate Reader Model 2390 (PerkinElmer). |
| Affinity data for this assay | |
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