This antibody gave a positive signal in HeLa whole cell lysate.
It also gave a positive signal in FFPE human skin tissue sections
IF/ICC: HeLa cell line
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The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Detects a band of approximately 49 kDa (predicted molecular weight: 49 kDa).
Use a concentration of 5 µg/ml.
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Expressed in a discontinuous manner in the basal cell layer of adult skin epidermis, but continuously in the basal layer of fetal skin epidermis and nail. Also expressed in the outer root sheath above the hair bulb in hair follicle (at protein level). Expressed homogeneously in all cell layers of the esophagus and exocervix, but detected in the basal cell layer only of oral mucosa, skin and in the basal plus the next two layers of the suprabasal epithelium of the palate.
ICC/IF image of ab111448 stained HeLa cells. The cells were 100% methanol fixed (5 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 (ab111448, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 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) at a concentration of 1.43µM. This antibody also gave a positive result in 100% methanol fixed (5 min) HepG2 cells at 5µg/ml, and in 4% formaldehyde fixed (10 min) HeLa and HepG2 cells at 5µg/ml.
Western blot - Anti-Cytokeratin 15 antibody (ab111448)
Anti-Cytokeratin 15 antibody (ab111448) at 1 µg/ml + HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate 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 : 49 kDa Observed band size : 49 kDa Additional bands at : 61 kDa. We are unsure as to the identity of these extra bands.
IHC image of Cytokeratin 15 staining in human normal skin formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab111448, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.