Surface Plasmon Resonance for Tubulin Binding Affinity Analyses

To evaluate the binding affinities of DJ95 with tubulin protein, we performed surface plasmon resonance analyses using a Biacore T200 system (GE Healthcare Life Sciences). A Series S Sensor Chip CM5 (GE Healthcare Life Sciences) was preconditioned with three consecutive 1-minute injections of 70% (w/w) BIA normalizing solution (GE Healthcare Life Sciences). Then, 20 μg/ml tubulin (Cytoskeleton Inc.) was immobilized to the sensor chip surface to attain 17,000 Resonance Units (1000 Resonance Units corresponds to an angle change of ∼0.1°). One of the four flow cells on the chip was left free as a negative control. DJ95, colchicine, or Combretastatin A4 (CA-4) (positive controls) was injected over the sensor chip surface for association analysis, followed by dissociation analysis. We adjusted the concentration gradients for each of the three compounds based on their different affinities to tubulin and different solubilities. The experiment data were obtained at 25°C with running a buffer PBS (10 mM phosphate, 2.7 mM KCl, and 137 mM NaCl), and 0.01% (v/v) Surfactant P20 (GE Healthcare Life Sciences) (pH 7.4). The flow rate was 30 µl/min. The analytes bound on the sensor chips were connected for 120 seconds and dissociated for 120 seconds. Regeneration of the sensor chips was performed for 30 seconds by 10 mM glycine-HCl buffer (pH 1.5). The equilibrium dissociation constant (K_d) was calculated by a steady-state fitting mode with Biacore T200 Evaluation Software, version 2.