2.8 Surface plasmon resonance assay
The binding affinity of natural compounds to STAT3 (127-722 AA) was evaluated by Surface Plasmon Resonance (SPR) assay using a Biacore T200 instrument (GE Healthcare, Piscataway, NJ, USA). After conditioning with 350 mM EDTA and 50 mM NaOH, 50 μg/mL of STAT3 protein was immobilized onto an NTA sensor chip at a flow rate of 10 μL/min in PBS containing 0.05% (v/v) Tween-20. The serial concentrations of natural compounds were injected into the flow system and analyzed, respectively. All measurements were performed in PBS with 0.05% (v/v) Tween-20, pH 7.4, at 25 °C. The STAT3 protein and natural compounds were allowed to associate for 90 s and then dissociate for 120 s. After dissociation, the sensor chip was washed with running buffer and regenerated with 350 mM EDTA and 50 mM NaOH. To avoid or reduce the influences of the bulk refractive index changes, data drift and injection noise, we performed the double reference subtractions prior to analysis. The binding affinity was measured by global fitting to a 1:1 Langmuir model using the Biacore Evaluation software (GE Healthcare).