| Assay Method Information | |
| | AF1-BD2 TR-FRET Binding Assay |
| Description: | His/Flag epitope tagged TAF1-BD21504-1635 was cloned, expressed, and purified to homogeneity. TAF1-BD2 binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound 1000 with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate, TAF1-BD2 (0.012 uM final) in 50 mM HEPES (pH 7.5; final), 50 mM NaCl (final), 1 mM TCEP (final), 0.1 mg/mL BSA (final), and 0.069 mM Brij-35 (final) was added either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After a 10 minute incubation at room temperature, biotin-ligand (0.04 uM final) was added. After 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6×His antibody (Perkin Elmer AD0110) and SureLight Streptavidin-Allophycocyanin (SA-APC, Perkin Elmer CR130-100) were added to a final concentration of 0.2 nM antibody and 0.025 uM SA-APC, respectively. After forty minutes of equilibration at room temperature, the plates were read on an Envision instrument and IC50s calculated using a four parameter logistic model (XLFIT). Compound 1000 and the TAF1-BD2 TR-FRET Binding Assay described above represent additional embodiments of the invention. |
| Affinity data for this assay | |
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