Purified RBP4 protein with >90% purity and 0.56 mg/mL concentration was immobilized on Biacore Sensor Chip CM5 (ITDD Biacore S200 Surface Plasmon Resonance instrument at University of Minnesota). The two-flow cell surface activated for using one as blank and other as test. Using Amine Coupling Kit (Cat. No. BR100050; Cytiva, Marlborough, MA, US) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), after surface activation, the purified RBP4 with immobilization buffer 10 mM Sodium acetate, pH 5.0, was immobilized with target of 1,200 Response Unit (RU) for achieving Rmax of 30RU in kinetic study. The reaction stopped and washed with Ethanolamine. The system was re-primed with running buffer PBST (phosphate-buffered saline solution with a 0.05% Tween20 detergent solution). The kinetic assay performed on the two flow cells, the blank was used as reference cell and the active cell with RBP4 was used for the binding study. The mouse and zebrafish RBPR2, mouse RBPR2 mutants affecting the “SYL” binding domain, and mouse STRA6 peptides (all containing the predicted RBP4 “SYL” binding residues) were chemically synthesized by Biomatik Corporation, Kitchener, ON, Canada. The peptides were serial diluted in running buffer with range of 0.8–26.6 μM and following parameter was run with contact time: 120 s, flowrate 30 μL/min, Dissociation time 300 s, Regeneration with Glycine-HCl, pH 2.5, contact time 30 s flowrate 30 μL/min and temperature 25°C. The program was run and non-specific binding on the reference cell subtracted bulk refractive index from the active sensorgram and analyzed for the association, dissociation and stabilization of the reads. The plot fitted with 1:1 binding program in Biacore™ Insight Evaluation Software, and the Graph, binding affinity plot, was plotted in GraphPad prism version 9.3.
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