125I-hepcidin binds to 2 unknown molecules in plasma and serum. (A) Human plasma (10 μL) or serum (10 μL) were incubated with 125I-human hepcidin (2.8 μM) for 1 hour at 37°C and 2 bands were detected ↓ corresponding to a complex of 125I-human hepcidin with unknown binding proteins. (B) Native fast-pressure liquid chromatography (FPLC) fractionation of blood plasma followed by (C) separation of fractions using native-gradient PAGE. FPLC fractions were trace labeled with 125I-hepcidin and separated by native gradient PAGE. After electrophoresis, the gel was vacuum dried, exposed to a storage phosphorimaging screen, and scanned on a phosphorimager. Results are typical of 3 or 4 experiments.