Figure 3.

Sequential affinity purification of placental hFcRn and HFE protein complexes and characterization of HFE-associated proteins. Lanes 1 and 3 illustrate blots of biotinylated hFcRn and associated proteins eluted from a human IgG Affigel-10 column. The flow-through from the human IgG Affigel-10 column was subjected to a second immunoaffinity purification step using immobilized CT16 antibodies. Lanes 2 and 4 contain blots of biotinylated proteins that were eluted from the CT16 IgG Affigel-10 column. On Western blots, the CT16 antibody did not cross-react with any polypeptide present in the hFcRn preparation (lane 1), but did identify a 48-kDa polypeptide band corresponding to the molecular weight of monomeric HFE protein in the fraction eluted from the CT16 IgG Affigel-10 column (lane 2). Western blot analysis using anti-human β₂M antibody (lanes 3 and 4) identified a 12-kDa polypeptide in both the hFcRn (lane 3) and HFE protein preparations (lane 4).