Human Scg3 (Sino Biological) was labeled with biotin using EZ-Link™ Sulfo-NHS-Biotin labeling reagent (NHS-Biotin, Thermo Fisher) according to the manufacturer’s protocol. Briefly, Scg3 in PBS was incubated with NHS-Biotin for 30 min at room temperature, followed by incubation with 1/10 volume of 100 mM Tris-HCl pH 8.0 to stop the reaction. Scg3-biotin was purified using desalt spin columns (Bio-Rad).
Epitope binning was performed to analyze epitope binding specificity of mAbs [18 (link), 19 (link)]. In brief, Scg3-biotin was incubated with streptavidin biosensors (Pall ForteBio). After washing, anti-Scg3 mAbs were incubated with Scg3 on biosensors as blocking mAbs, followed by washing and incubation with different anti-Scg3 mAbs using Octet RED96 instrument (Pall ForteBio) to monitor binding kinetics.
Epitope binning was performed to analyze epitope binding specificity of mAbs [18 (link), 19 (link)]. In brief, Scg3-biotin was incubated with streptavidin biosensors (Pall ForteBio). After washing, anti-Scg3 mAbs were incubated with Scg3 on biosensors as blocking mAbs, followed by washing and incubation with different anti-Scg3 mAbs using Octet RED96 instrument (Pall ForteBio) to monitor binding kinetics.
