Lance eu w1024 streptavidin

A total of 9.8 μl N-terminal Avi Tag-WRN(Asn517–Pro1238) in 30 mM Tris-HCl pH7.5, 1 mM MgCl₂, 30 mM NaCl, 0.02% BSA, 0.1% Pluronic F127 was incubated with 200 nl compound for 45 min at room temperature in white 384-well plates (Greiner Bio-One, 781207). Then, 10 μl Cy5-labelled ATPγS (EDA-ATPγS-Cy5, Jena Bioscience, NU-1609-CY5) and europium-labelled Streptavidin (LANCE Eu-W1024 Streptavidin, Perkin Elmer, AD0063) were added. The final concentrations in the assay plate were 15 nM WRN, 10 nM ATPγS-Cy5 and 2 nM Eu-Streptavidin. With the TECAN infinite M1000 PRO, Eu was excited at 340 nm and the fluorescence emission was measured at 620 nm (F₆₂₀) and 665 nm (F₆₆₅) after 30 min. A ratio of F₆₆₅ over F₆₂₀ was then calculated to determine the FRET efficiency in each well. This assay protocol was used during hit optimization to build the initial structure–activity relationship and was also adapted to 1,536-well plates for screening purposes.

Biotinylated N-Avitagged-TEAD4^217–434 (1 nM) and LANCE Eu-W1024 Streptavidin (0.5 nM, PerkinElmer) were pre-incubated for 1 h at room temperature in 50 mM HEPES pH 7.4, 100 mM KCl, 0.05% (v/v) Tween-20, 0.25 mM TCEP, 1 mM, and 0.05% (w/v) BSA. Different YAP mutants (20 nM) and serial dilutions of them were added and incubated in white 384-well plates (Greiner Bio-One International) for 1 h at room temperature. DMSO was present at 2% in the assay. The solubility of the peptides in assay buffer was measured by dynamic light scattering with a Dyna Protdevice (Wyatt technology Corp.). The fluorescence in the TR- FRET assay was measured with a Genios Pro reader (Tecan) (50 μs delay between excitation and fluores- cence, 75 μs integration time, excitation wavelength 340 nm, emission wavelengths 620 and 665 nm). Data analyses were carried out using the TR-FRET 665/620 nm emission ratio. The IC₅₀ values were obtained by nonlinear regression analysis with GraphPad Prism (GraphPad Software) (Bokhovchuk et al., 2020a (link)).