Fluorescence polarization assay

Apparent peptide dissociation constants were determined based on fluorescence polarization (FP) measurements performed on a POLARstar Omega microplate reader (BMG Labtech) using FITC-labeled peptides as described previously [20]. Titrations were performed using serial dilutions of xlKu80vWA in a mixture with a final concentration of 100 nM FITC-labeled peptide in FP buffer (25 mM HEPES, 150 mM NaCl, 1 mM EDTA, 2 mM DTT, 0.05 % Tween20, 0.1 % BSA, pH 7.4). 50 µL volumes of the binding reactions were pipetted into wells of a 96-well black flat-bottomed plate (Corning, ME) and fluorescence polarization was read at room temperature, using excitation at 485 nm and emission at 520 nm. Data for the interaction of a peptide with xlKu80vWA were fit to a single-site binding equation as described previously [20]. All experiments were at least triplicated and data represent the mean. Fluorescein-labeled peptides are listed in Table S1.