A Stu2 CTS peptide (855-888) with a C-terminal cysteine, synthesized by the Tufts University Core Facility, was labeled with Oregon Green maleimide (Thermo Fisher) according to the manufacturer's instructions. The labeled peptide was separated from unreacted dye, first by cation exchange chromatography with Source 15S resin (Cytiva), followed by gel filtration chromatography on a Superdex 75 column (Cytiva). Fluorescence polarization was measured with a SpectraMax plate reader (Molecular Devices) at 25°C with excitation and emission wavelengths set at 484 and 525 nm, respectively. Binding of 50 nM Oregon Green CTS to 10 µM Ndc80cdwarf was carried out in buffer containing 10 mM HEPES pH 7.0 and 100 mM NaCl, adding increasing concentrations of unlabeled Stu2 CTS peptide. Measurements were carried out in duplicate. Curve fitting used a single site saturation binding model implemented in GraphPad Prism 9.

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