Affinity Purification of C3 Complement Protein

Pull-downs used to identify C3 (Fig. 1c) were performed as previously described^{22 (link)}. In brief, glutathione-sepharose 4B (GE Healthcare, 140 μl slurry/pull-down) was incubated with GST-tagged protein (1 mg/pull-down) for 30 min, washed with PBS and incubated with bovine serum (Sigma-Aldrich, 1 ml/pull-down) or a PBS control for 60 min. ISG65 E1125A produced in E. coli was used in these experiments. The beads were rapidly washed with three PBS washes with a total time of 5 min. Bound protein was eluted by adding SDS-polyacrylamide gel electrophoresis sample buffer and heating at 50 °C. The pull-downs using GST-ISG65A as bait were performed in three replicates. Pull-downs used to identify C3 from different mammalian species (Fig. 1d) were performed by binding avi-tagged ISG65 E1125G to Pierce Streptavidin Agarose beads (Thermo Fisher). Serum from various mammalian species (Sigma) was added to ISG65-streptavidin beads via gravity flow. The beads were then washed three times with HBS pH 7.4, and C3 bound to ISG65 eluted with 0.1 M sodium acetate pH 4, 500 mM NaCl.