Pbs ep buffer

Binding experiments were carried out using Biacore S200 SPR sensors with control software version 3.0 and Sensor Chip CM5 (carboxymethylated dextran surface). All assays were carried out at 25°C. For the pre-binding experiment, pure RXRα-LBD protein was dissolved with acetate buffers with different pH values (pH4.0, pH4.5, pH5.0, and pH5.5) and flowed through the surface of the chip at a rate of 5 μL/min. Then, the chip surface was activated following a standard EDC/NHS protocol with Biacore PBS-EP buffer used as the running buffer. RXRα-LBD protein at a concentration of 0.4 g/L in 10 mM phosphate buffer pH 5.0 was then injected for 12 min followed by a 7- min injection of 1 M ethanolamine to inactivate residual active groups. Typically, approximately 2000 RU receptor protein was immobilized per flow cell. By reference to the coupling steps of RXRα-LBD protein, the protein was coupled to the chip. For the preparation of the α-Mangostin solution, the insoluble residue was pelleted by centrifugation and discarded. α-Mangostin was injected into the protein channel and blank channel at seven concentrations (0.625 nM, 1.25 nM, 2.5 nM, 5 nM, 10 nM, 20 nM, and 40 nM). The supernatant (200 μL) was injected at a flow rate of 20 μL/min. The protein binding period was set to 3 min, and the dissociation period was set to 300 s. The chip was regenerated with glycine-HCl (pH 2.5, 10 mM).