Biacore t200 model

All chemicals and reagents were purchased from Sigma unless stated otherwise. The PCR reagents, restriction enzymes and B-PER reagent were obtained from Thermo Scientific. The cysteine-alkyne bifunctional linker (Figure S1A, Supplementary Materials) was purchased from Eurogentec, the azido-propylamine linker from Jena bioscience (Figure S1B, Supplementary Materials) and the N-hydroxysuccinimide (NHS) derived ester linker 2,5-dioxopyrrolidin-1-ylhex-5-ynoate (Figure S1C, Supplementary Materials) was self-synthesized according to Jagadish et al. [55 (link)]. The pMXB10 vector, E. coli SHuffle^® T7 competent cells and chitin resin were purchased from New England Biolabs. The two recombinant human VCAM1 antigens (hVCAM1) were bought from R & D Systems (MW of 270 kDa) and Peprotech (MW of 180 kDa) while the recombinant mouse VCAM1 (mVCAM1) antigen was purchased from Bioconnect (Huissen, The Netherlands, MW of 95 kDa). The ellipsometer and silicon wafers were bought from Synapse B.V. (Maastricht, The Netherlands) and the Biacore™ (Diegem, Belgium) C1 sensor chips from GEHealthcare. The SPR experiments were performed with a Biacore™ T200 model (GE Healthcare).

The surface plasmon resonance (SPR) assay was measured using the BIAcore T200 model (GE Healthcare, Chicago, IL, USA) with HBS-EP buffer (1×HBS-EP, GE Healthcare, Chicago, IL, USA) containing 5% dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA). The pH scouting for IL-6 (PeproTech, Rocky Hill, NJ, USA), IL-6Rα (PeproTech, Rocky Hill, NJ, USA), and gp130 (Sino Biological, Beijing, China, or ANRT, Daejeon, Korea) immobilization was performed in 10 mM acetate buffer at pH 4.5. IL-6, IL-6Rα, and gp130 were immobilized on a CM5 chip at 750, 1200, and 2000 response units (RU). Butein was injected into the IL-6, IL-6Rα and gp130-immobilized flow cell at concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μM with a 25 μL/min flow rate for 150 s and allowed to dissociate for 300 s. Steady-state KD was determined via the T-200 BIAevaluation software (GE Healthcare, Chicago, IL, USA).