Biacore s200 evaluation software version 1

Surface plasmon resonance studies were carried out using a Biacore S200 instrument (GE Healthcare, Uppsala, Sweden) at 25°C. G6PDH protein was immobilized on the Series S CM5 Sensor Chip using the standard amine-coupling method. G6PDH protein was diluted to 50 μg/mL in 10 mM sodium acetate buffer, pH 4.5. Immobilization was performed using an amine-coupling kit (GE Healthcare) following the manufacturer’s protocol. The kinetics and affinity assay were determined at a flow rate of 30 μL/min using PBS-P buffer [20 mM phosphate buffer, 2.7 mM KCl, 137 mM NaCl, and 0.05% (v/v) P20 surfactant]. Diluted caffeine, NADP⁺, and G6P were stored at 4°C and placed into the rack tray before injection. The association and dissociation times were both 90 s. The K_D values were calculated with the kinetics and affinity analysis option of Biacore S200 Evaluation Software Version 1.1 (GE Healthcare).

The binding affinity of MIC-1 and PTP1B (Sino Biological, Cat. 10,304-H07E) was determined with a Biacore S200 system (GE Healthcare, Uppsala, Sweden). PTP1B was immobilized on the Fc2 channel of the CM5 chip using an amine-coupling kit (GE Healthcare) following the manufacturer’s protocol. PTP1B was diluted to 100 μg/ml with a 10 mM sodium acetate solution (pH 4.0). Kinetics and affinity were determined at a flow rate of 30 μl/min using PBS-P buffer. MIC-1 association and dissociation rates were measured for 90 s each. The KD values were calculated using the kinetics and affinity analysis option of the Biacore S200 Evaluation Software Version 1.1 (GE Healthcare).