| Assay Method Information | |
| | Polybromodomain-1 BromoDomain-5 (PB1-BD5) SM TR-FRET Binding Assay |
| Description: | Histidine-Flag-PB1-BD5 Bromodomain645-766 (S645-D766; Swiss Prot Q86U86; mhhhhhhasdykddddkgslvpr\gsSGISPKKSKYMTPMQQKLNEVYEAVKNYTDKRGRRLSAI FLRLPSRSELPDYYLTIKKPMDMEKIRSHMMANKYQDIDSMVEDFVMMFNNACTYNEP ESLIYKDALVLHKVLLETRRDLEGD SEQ ID NO:3) was cloned, expressed, and purified to homogeneity. PB1-BD5-BD binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule ligand (Example 248) with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate, His-Flag-PB1-BD5-BD (1 nM final) was combined with biotinylated-ligand (30 nM final) in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 20 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6×His antibody ( 6×His disclosed as SEQ ID NO: 4) (Perkin Elmer AD0110) and SureLight Streptavidin-Allophycocyanin (SA-APC, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nM antibody and 50 nM SA-APC, respectively. After sixty minutes equilibration, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear fit. |
| Affinity data for this assay | |
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