| Assay Method Information | |
| | In-Vitro Biochemical Assays |
| Description: | Avi-humanTEAD4217-434 (1 nM, produced as described in Hau et al. ChemBioChem 14, 1218, 2013) and LANCE Eu-W1024 Streptavidin (0.5 nM, PerkinElmer) were first pre-incubated for 1 h at room temperature in HEPES (pH 7.4, 50 mM), KCl (100 mM), Tween-20 (0.05%), TCEP (0.25 mM), EDTA (1 mM), and BSA (0.05%)]. N-terminus Cy5 labeled humanYAP60-100 (20 nM) was then added to this preparation. Compounds were dissolved at 10 mM in 100% DMSO and serial dilutions were made in 100% DMSO. The diluted compound solutions were incubated in white 384-well plates (Greiner Bio-One) for 1 h at room temperature with the above described mix. The final DMSO concentration present in the assay was 1%. The fluorescence was measured (50 μs delay between excitation and fluorescence, 75 μs integration time) with a Genios Pro reader (Tecan) and use of an excitation wavelength of 340 nm and emission wavelengths of 620 nm and 665 nm. Data analyses were carried out by using the TR-FRET ratio emission 655 nm/620 nm. The IC50 values were estimated by fitting the data by nonlinear fit regression (GraphPad Prism). In the alternative format, the assay was conducted in the presence of 5 nM His-humanTEAD4217-434, 10 nM N-biotinylated YAP60-100, 0.2 nM anti-His Europium labelled antibody and 10 nM SA-XL665. |
| Affinity data for this assay | |
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