Serum ferritin is a fully assembled 24-subunit polymer. (A) Gel filtration of mouse serum on a Sephacryl S-300 26/60 column using an Akta basic fast-protein liquid chromatography (FPLC) machine (Pharmacia). Absorbance was measured at 280 nm for protein (light grey curve) and 420 nm suggestive for iron (dark grey curve). Note the different scales for 280 nm and 420nm. Absorbance maxima at 420 nm were found at 105 to 110 and 135 to 140 mL, suggesting the presence of ferritin in these fractions. A large peak with absorbance at 280 nm at 170 to 175mL corresponds to non–iron-containing irrelevant serum proteins (1 representative experiment is shown of 3). (B) Ferritin quantification by enzyme-linked immunosorbent assay (ELISA). (C) Purified mouse liver ferritin was loaded onto the same column and eluted as a single peak at 135 to 140 mL. Absorbance was measured at 280 nm for protein (light grey curve), 260 nm for nucleic acid contamination (not shown), and 420 nm suggestive for iron (dark grey curve). (D) Calibration curve obtained with molecular weight markers for gel filtration chromatography (Sigma-Aldrich) run on the same column.