1 µM of Xyn-Doc fusion proteins (wild-type Q1, Q3, QQ Doc fusions) bearing either wild-type or mutant Doc domains were adsorbed onto surfaces of the wells of a 96-well nunc maxi sorp plate (Thermo Scientific, Pittsburgh, PA). After blocking (2% (w/v) BSA, 0.05% Tween 20 in TBS buffer) and several rinsing steps, a red fluorescent protein-cohesin (StrepII-TagRFP-Coh2 (C.t.), Addgene ID 58,710 (Otten et al., 2014 (link))) fusion construct was incubated to the unspecifically immobilized Doc fusion proteins over a range of concentrations. After further rinsing, the fluorescence of the TagRFP domain was measured with a multi-well fluorescence plate reader ( M1000 PRO, Tecan Group Ltd., Männedorf, Switzerland). Fluorescence values were plotted against their corresponding concentration values for each protein variant, and 4 parameter logistic nonlinear regression model functions were fitted to the data to determine the transition point of the curve.
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