Streptavidin coated chip

SPR experiments were conducted in duplicate or triplicate using a BiaCore T200 instrument (Cytiva) in SPR buffer [150 mM NaCl, 20 mM sodium phosphate (pH 7.4), and 0.1% Tween 20]. Approximately 1000 resonance units of biotinylated hTAPBPR or chTAPBPR or hTAPBPR mutants were immobilized at 10 μl/min on a streptavidin-coated chip (GE Healthcare). Various concentrations (0, 0.05, 0.1, 0.25, 0.5, 0.75, 1, 2.5, 5, 7.5, 10, and 20 μM) of HLA molecules were selected and injected over the chip at 25°C at a flow rate of 30 μl/min for 60 s, followed by a buffer wash with 180-s dissociation time. For MR1 experiments, various concentrations (0, 5, 10, 15, 20, 25, 50, and 100 μM) were selected and used on hTAPBPR WT and mutants or chTAPBPR immobilized streptavidin chip. Before SPR experiments of UV-irradiated molecules, HLA molecules were diluted to the desired concentrations and UV-irradiated for 20 min at 365 nm. SPR data were then immediately acquired following a 20-min UV irradiation. The SPR sensorgrams, association/dissociation rate constants (k_a and k_d), and equilibrium dissociation constants (K_D) were analyzed using the surface-bound analysis settings in BiaCore T200 evaluation software (Cytiva) or fitted using one site-specific binding by GraphPad Prism v9. SPR sensorgrams and saturation curves were prepared in GraphPad Prism v9.