| Assay Method Information | |
| | TEAD4 FRET Assay |
| Description: | Compound IC50 values were assessed following a 10-point, half-log10 dilution schema starting at 100 μM compound concentration. Specifically, human TEAD protein from TEAD4(217-434) was cloned into an overexpression vector, expressed as an N-terminal HIS-TEV-Avi-tagged fusion protein in E coli, and subsequently purified, then protein was chemically depalmitoylated & biotinylated. The assay was performed in 384-well LV plates (384-well black, medium binding, PS, HIBASE, GREINER #784076) and run in the presence and absence of the compound of interest. Each well of 5 μL assay mixture contained 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.05 mM EDTA, 1 mM TECP, 1% DMSO, 0.03% Pluronic acid F127, 20 nM dePal Avi TEAD4 (217-434)-depalmitoylated & biotinylated protein, 0.8 nM Streptavidin Terbium cryptate (CisBio #610SATLB), 625 nM FAM labelled Probe A. Reactions were incubated at 25° C. for 120 min before reading on a PHERASTAR FSX Plate Reader (337 520 490 HTRF module required) (Supplier BMP). |
| Affinity data for this assay | |
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