High-throughput Enrichment of C3 from Serum

C3 was isolated from the blood serum samples by Concanavalin A lectin affinity chromatography as described previously (10 (link)). In brief, C3 was enriched from 10 μl of serum in a high-throughput manner, using Concanavalin A-Sepharose 4B (Global Life Sciences Solutions, Marlborough, MA, United States) resin slurry placed into 96-well polypropylene filter (Orochem Technologies Inc., Naperville, IL, United States). The conditioned resin was loaded with serum samples that had been diluted 10-fold with binding buffer (20 mM TRIS pH 7.4, 0.5 M NaCl, 1 mM CaCl2, MnCl2), and the mixture was incubated while shaking overnight at 4°C. A vacuum manifold (Pall Corp., NY, USA) was used for washing of the lectin matrix. Afterwards, glycoproteins were eluted with elution buffer (200 mM methyl α-D-mannopyranoside (Sigma-Aldrich, St. Louis, MO, United States) in 0.1M acetic acid (Merck KgaA, Darmstadt, Germany), pH 3.0)) by low-speed centrifugation. The resulting eluates were then dried down immediately in a SpeedVac Vacuum Concentrator (Thermo Scientific, Waltham, MA, United States).