Surface plasmon resonance studies

To study biomolecular interaction between integrin and 25HC in real-time, SPR was performed using a Biacore 3000 instrument (GE Healthcare, Piscataway, New Jersey) according to the manufacturer’s instructions. Purified human αvβ3 integrin protein (Yo proteins AB, Huddinge, Sweden) was covalently immobilized on a flow cell of CM5 chip (carboxy-methylated dextran coated) in 10 mM sodium acetate buffer, pH 4.0, using EDC/NHS amine coupling chemistry at 25 °C. The unused dextran surface was then inactivated by injecting 1 M Ethanolamine, pH 8.5. Similarly, the blank control flow cell was simultaneously activated and inactivated without the protein for background subtraction of any non-specific response. For kinetic analyses, increasing concentrations of 25HC (0 nM, 16 nM, 40 nM, 160 nM, 640 nM, 1.6 µM) in the running buffer HBS-P (10 mM HEPES, 0.15 M NaCl, 0.005% polysorbate 20, pH 7.4) with 1% DMSO were injected at a flow rate of 20 μl/min for 150 s. Following dissociation, the chip surface was regenerated with the running buffer. The background subtracted SPR sensorgrams were quantitatively evaluated to determine K_D (apparent affinity constant) by using the Biacore 3000 Evaluation Software (GE Healthcare) and the Langmuir 1:1 binding model.