Hepcidin (Hepc, hepatic bactericidal protein) or LEAP (liver expressed antimicrobial peptide) is small, cysteine-rich peptide, antimicrobial peptide similar to defensins and thionins. Hepcidin (unprocessed, proprotein in mouse 83 aa, rat/human 84 aa) are almost exclusively produced in liver. Human hepcidin is produced from 84-aa precursor, including a putative 24-aa signal peptide. The signal peptide is cleaved to produce pro-HEPC, which is further processed to generate the mature secreted form of hepcidin (C-terminal 20, 22 or 25-aa HEPC). In humans, 20-aa and 25-aa appears to be the major HEPC secreted peptides with antimicrobial activities. The three secreted HEPC alternatively spliced HEPC peptides differ at the N-terminus.
The link between hepcidins and iron metabolism is that hepcidin expression is abolished in mice exhibiting iron-overload due to the targeted disruption of USF2 (upstream stimulatory factor 2) gene resembling the situation in hfe-/- mice. The human gene is located at chromosome 19, in close proximity with Usf2 gene. Hepcidin levels are increased in iron loading and in beta-2 microglobulin knockout mice. Hepcidins are devoid of IRE. Like other antimicrobial peptides, hepcidin is up-regulated by lipopolysaccharides (LPS).
Applications
Suitable for use in ELISA, Western Blotting. Other applications not tested.
Recommended Dilution
Western Blot: 1:1000-1:5000 for antiserum and 1-10ug/ml for aff. pure IgG using ECL. ELISA: ~1:100,000 using 50-100ng control peptide/well. Optimal dilutions to be determined by the researcher.
Control peptide, because of the small size (2-3kD), is not recommended for Western. It should be used in ELISA or antibody blocking (use 5-10ug control peptide per 1ug of IgG or 1ul of antiserum) experiments to demonstrate antibody specificity. Full length, human 25-aa, 20-aa HEPC and mouse 25-aa HEPC are also available for various studies.
Storage and Stability
May be stored at 4°C for short-term only. For long-term storage and to avoid repeated freezing and thawing, aliquot and add glycerol (40-50%). Freeze at -20°C. Aliquots are stable for at least 12 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
Immunogen
An 11-aa peptide sequence from the N-terminus (after the signal peptide) of human Pro-HEPC was synthesized, conjugated to KLH.
Form
Supplied as a liquid, neat serum, 40% glycerol.
Specificity
The human sequences is 63% conserved in mouse/rat pro-HEPC. This sequence is removed from the mature 20-25 secreted forms of HEPC. So antibodies will not react with the secreted forms of HEPC.