Hepcidin-binding protein in plasma is supershifted with an anti–α2-M antibody. (A) Human plasma was incubated with 125I-human hepcidin (2.8 μM) for 1 hour at 37°C. This sample was then divided into portions to which increasing amounts of anti–α2-M antibody (5-20 μL; concentration 200 μg/mL) or anti–cyclin D1 antibody (20 μL; concentration 200 μg/mL) were added. The samples were separated using native-gradient PAGE. After electrophoresis, the gel was vacuum dried, scanned, and analyzed using a phosphorimager. Results are typical from 3 separate experiments. (B) The radioactivity in the 125I-hepcidin–α2-M protein band (▴) and the sample wells (■) were plotted to show that the addition of anti–α2-M antibody progressively decreases the radioactivity in the 125I-hepcidin–binding protein band and increases the activity in the sample wells. The activity in the wells is because the high molecular weight antibody–α2-M complex cannot penetrate into the gel. The radioactivity in the sample well with no antibody is caused by the presence of cationic free hepcidin, which does not migrate into the gel. Results are typical of 3 separate experiments.
