| Assay Method Information | |
| | TAF1-BD1 TR-FRET Binding Assay |
| Description: | His/Flag epitope tagged TAF1-BD11381-1497 was cloned, expressed, and purified to homogeneity. TAF1-BD 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-BDI (0.08 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 (0.08 uM final) was added. After 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6×His antibody (Perkin Elmer ADO 110) and SureLight Streptavidin-Allophycocyanin (SA-APC, Perkin Elmer CR130-100) were added to final concentrations of 0.2 nM antibody and 0.05 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). The TAF1-BD1 TR-FRET Binding Assay described above represents an additional embodiment of the invention. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |