Anti-Hepcidin-25 antibody (ab75883)

Western blot - Hepcidin-25 antibody (ab75883)

Anti-Hepcidin-25 antibody (ab75883) at 1 µg/ml + Human HAMP Full-length Recombinant Protein (Tagged) at 0.1 µg

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size : 3 kDa
Observed band size : 32 kDa (why is the actual band size different from the predicted?)


ab75883 was tested against Human HAMP Full-length Recombinant Protein (Tagged) predicted to run at 32kDa.

Immunocytochemistry/ Immunofluorescence - Hepcidin-25 antibody (ab75883)

ICC/IF image of ab75883 stained HeLa cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal Goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab75883, 1µg/ml) overnight at +4ºC. The secondary antibody (green) was Alexa Fluor® 488 Goat anti-Rabit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue). This antibody also gave a positive result in 4% PFA fixed (10 min) HEK293, HepG2, and MCF-7 cells at 1µg/ml.

Immunocytochemistry/ Immunofluorescence - Hepcidin-25 antibody (ab75883)

ICC/IF image of ab75883 stained HepG2 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal Goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab75883, 5µg/ml) overnight at +4ºC. The secondary antibody (green) was Alexa Fluor® 488 Goat anti-Rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue). This antibody also gave a positive result in 4% PFA fixed (10 min) HeLa, MCF-7 cells at 5µg/ml, and in 100% Methanol fixed (5 min) Hek293, HepG2, and MCF-7 cells at 5µg/ml.

Immunohistochemistry (Frozen sections) - Hepcidin-25 antibody (ab75883)

ab75883 staining Hepcidin-25 in Mouse liver tissue sections by Immunohistochemistry (IHC-Fr - frozen sections). Tissue was fixed with formaldehyde, permeabilized with 0.1% Triton X 100 and blocked with 5% serum for 1 hour at 25ºC. Samples were incubated with primary antibody (1/250 in PBS Tween) for 12 hours at 4ºC. An Alexa Fluor^®594-conjugated goat anti-rabbit IgG polyclonal (1/250) was used as the secondary antibody. Nuclei were stained with DAPI.

This image is courtesy of an anonymous Abreview