Western blot (WB) using novel monoclonal antibodies (mAb; 4G2, 7D4, 10B6) in comparison with anti‐C5 mAb RO7112689. Proteins; human C5, C5b6 and C6 (1 μg) were resolved on 4–20% PAGE gels under non‐reducing (NR) and reducing (R; 5% β‐mercaptoethanol) conditions. Blots were probed with mAb 4G2 (a), 7D4 (b), 10B6 (c), RO7112689 (d). All mAb detected intact C5 and C5b but not C6 in the C5b6 complex. The mAb 4G2 and 7D4 bound the C5α chain, while mAb 10B6 and RO7112689 bound the C5β chain. All mAb except 4G2 detected C5b6 in reducing conditions. In‐house produced C5b6 probed with RO7112689 showed trace contaminating human antibody bands from the immunoaffinity purification process detected by the anti‐human IgG secondary. Results are representative of multiple analyses.