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).