Ab17931

C9-depleted serum and purified C9 standard were generated using affinity chromatography. Human serum was obtained from the Australian Red Cross Blood Service under QIMRB human ethics approval P2352. Serum from five healthy donors was pooled, diluted 1:1 with PBS then sterile filtered (0.22 µm). A 20 mL sample of the pooled serum was injected onto a 1 mL PBS-equilibrated HiTrap NHS-Activated HP affinity column (Cytiva, North Ryde, Australia) coupled to a monoclonal anti-C9 antibody [26 ]. C9-depleted serum fractions were collected in the flow-through and depletion confirmed by ELISA before pooling. The column was thoroughly washed with PBS before C9 was eluted with 5 mL of 0.1 M glycine, pH 2.5 and neutralized by addition of 0.5 mL of 1.5 M Tris, pH 8.8. Eluted C9 was desalted and buffer exchanged vs. PBS using a Zeba 7 K MWCO spin desalting column (Thermo Fisher Scientific) and concentrated using an Amicon ultra 10 K MWCO centrifugal filiter unit (Merck Millipore, Bayswater, Australia). C9 purity was assessed by SDS-PAGE and Western blot using a commercial anti-C9 antibody (ab17931, Abcam, Melbourne, Australia). Protein concentration of serum purified C9 was determined by BCA assay (Thermo Fisher Scientific).