The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
Use a concentration of 1 µg/ml. Detects a band of approximately 20 kDa (predicted molecular weight: 23 kDa).
Use at an assay dependent concentration.
May be involved in macrophage-mediated cellular proliferation. It is mitogenic for fibroblasts and smooth muscle but not endothelial cells. It is able to bind EGF receptors with higher affinity than EGF itself and is a far more potent mitogen for smooth muscle cells than EGF. Also acts as a diphtheria toxin receptor.
Contains 1 EGF-like domain.
Several N-termini have been identified by direct sequencing. The forms with N-termini 63, 73 and 74 have been tested and found to be biologically active. O-linked glycan attachment sites were determined by Edman degradation, O-glycanase digest suggests mucin-type glycosylation (done in HB-EGF purified from histiocytic lymphoma cell line U-937).
Cell membrane and Secreted > extracellular space. Mature HB-EGF is released into the extracellular space and probably binds to a receptor.
ICC/IF image of ab92620 stained MG63 cells. The cells were 4% formaldehyde 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 ab92620 at 5µg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor® 488 goat anti- rabbit (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.
Western blot - HB EGF antibody (ab92620)
Anti-HB EGF antibody (ab92620) at 1 µg/ml + Human stomach tumor tissue lysate (adenocarcinoma) - total protein (ab29678) at 10 µg
Secondary Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution Developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 23 kDa
Exposure time : 4 minutesThe band observed at 20 kDa could potentially be a cleaved form of HB EGF due to the presence of a 19 amino acid signal peptide.
Altonsy MO et al. Context-dependent cooperation between nuclear factor ?B (NF-?B) and the glucocorticoid receptor at a TNFAIP3 intronic enhancer: a mechanism to maintain negative feedback control of inflammation. J Biol Chem289:8231-9 (2014).
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