Human Hepcidin protein, His and Human IgG1 Fc tag

Hepcidin (Hepc) is a regulator of iron metabolism. Hepcidin inhibits iron transport by binding to the iron export channel ferroportin which is located on the basolateral surface of gut enterocytes and the plasma membrane of reticuloendothelial cells (macrophages). Hepcidin ultimately breaks down the transporter protein in the lysosome. Inhibiting ferroportin prevents iron from being exported and the iron is sequestered in the cells. Besides, Ferroportin (FPN) has been identified as an interactor of Hepc, thus a binding ELISA assay was conducted to detect the interaction of recombinant human Hepc and recombinant human FPN. Briefly, Hepc were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to FPN-coated microtiter wells and incubated for 2h at 37ºC. Wells were washed with PBST and incubated for 1h with anti-Hepc pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37ºC. Finally, add 50 μl stop solution to the wells and read at 450nm immediately. The binding activity of Hepc and FPN was in a dose dependent manner.