Confirmation that albumin is a second hepcidin-binding protein that binds this ligand nonspecifically. (A) Whole human plasma and albumin (purity: 99%) were separated using native PAGE and stained with Coomassie blue for protein. Note that the band of purified albumin comigrates with the most abundant protein present in human plasma (albumin). (B) Whole plasma and purified albumin were trace labeled with ¹²⁵I-hepcidin, resolved by native PAGE, and visualized using a phosphorimager. The band of purified albumin-¹²⁵I-hepcidin comigrates with the bottom band present in human plasma (albumin), which also binds ¹²⁵I-hepcidin. The top band in lane 1 is ¹²⁵I-hepcidin bound to α₂-M. (C) Increasing amounts of ¹²⁵I-hepcidin (up to 1400 nM) were added to albumin (1.5 nM), the mixture was incubated for 1 hour at 37°C, separated by native PAGE, and the albumin–¹²⁵I-hepcidin complex was visualized and quantified using a phosphorimager. Results in panels A and B are typical of 3 experiments while those in panel C are mean ± SD of 3 experiments.