Semiwet transfer

For pull-down assay, 0.1 mg/mL of purified protein was immobilized on Pierce NHS-activated magnetic beads (ThermoFisher) following the manufacturers’ instructions. The beads were incubated with 10 mM EDTA and 50 μL serum (21 °C, 30 min). The beads were washed 3 times with 1 mL PBS + 0.05% Tween20, once with 100 μL PBS, and boiled in 50 μL SDS- PAGE loading buffer. The eluted proteins were separated on an SDS polyacrylamide gel and observed by Coomassie staining, silver staining, or Western blotting. For blotting the SDS/PAGE-separated proteins were transferred to a PVDF membrane (Amersham Hybond P0.2 PVDF, 55 GE) by semiwet transfer (Bio-Rad) and blocked for 1 h with 2% milk. Primary antibody (α-C5: 1:80,000, Complement Technology). Secondary antibody (α-goat HRP, Promega, 1:10,000). The blot was developed using ECL Western Blotting Substrate (Promega) and imaged using Amersham Hyperfilm ECL (GE Healthcare).

For blotting the SDS/PAGE-separated proteins were transferred to a PVDF membrane (Amersham Hybond P0.2 PVDF, 55 GE) by semiwet transfer (Bio-Rad) and blocked for 1 h with PBS/2% milk.
Primary antibodies were purchased from CompTech (Complement Technologies Inc, USA): goat α-properdin, 1:2,000, goat α-Factor B, 1:4000, goat α-Factor D, 1:250. Secondary antibody (donkey α-goat HRP, Promega, 1:10,000). For His-tagged proteins, the Penta-His HRP Conjugate Kit (Qiagen) was used following the manufacturer’s instructions.
Blots were developed using ECL Western Blotting Substrate (Promega) and imaged using Amersham Hyperfilm ECL (GE Healthcare) or using a ChemiDoc XRS + imaging system (Biorad).