FG binding to HZ under physiologic conditions. (A) FG binding to FG-free HZ. FG-free HZ, obtained from Albumax-supplemented parasite cultures (lane 3), was incubated in undiluted fresh plasma for 5 and 30 minutes (lane 1 and lane 2, respectively), or in undiluted fresh serum from the same healthy donor (lane 4). After extensive washings, proteins extracted from HZ (corresponding to 100nmol heme in terms of heme content) were separated by 10% SDS-PAGE and stained with Coomassie R250. Plasma-proteins (PL; 15 μg) or FG run as standard (lane 5 and lane 6, respectively). (B) Western blot of proteins extracted from the same samples of panel A (corresponding to 50nmol heme/lane) were separated by 10% SDS-PAGE, transferred to nitrocellulose, and probed with a mouse monoclonal anti-FG γ-chain and a peroxidase-linked secondary Ab. FG γ-chain was detected by ECL. PL (15 μg) or solubilized FG was used as standard. (C) FG binding to BH. BH (corresponding to 100nmol heme in terms of heme content) was incubated and separated by SDS-PAGE as indicated for FG-free HZ and silver stained. Representative blots selected from 4 with similar results are shown.