Purification notesPrior to conjugation, the antibody was purified by Protein A affinity chromatography. The purified antibody is conjugated with Fluorescein isothiocyanate (FITC) under optimum conditions. The reagent is free of unconjugated FITC.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use 10µl for 106 cells. (in a 100µl cell suspension)
ab91356-Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.
Use a concentration of 5 µg/ml.
FunctionInvolved in the uptake of thrombin-antithrombin complexes by hepatic cells (By similarity). When phosphorylated, plays a role in filament reorganization. Involved in the delivery of mutated CFTR to the plasma membrane. Together with KRT8, is involved in interleukin-6 (IL-6)-mediated barrier protection.
Tissue specificityExpressed in colon, placenta, liver and very weakly in exocervix. Increased expression observed in lymph nodes of breast carcinoma.
Involvement in diseaseDefects in KRT18 are a cause of cirrhosis (CIRRH) [MIM:215600].
Sequence similaritiesBelongs to the intermediate filament family.
Post-translational modificationsPhosphorylation at Ser-34 increases during mitosis. Hyperphosphorylated at Ser-53 in diseased cirrhosis liver. Phosphorylation increases by IL-6. Proteolytically cleaved by caspases during epithelial cell apoptosis. Cleavage occurs at Asp-238 by either caspase-3, caspase-6 or caspase-7. O-glycosylated at multiple sites; glycans consist of single N-acetylglucosamine residues.
Overlay histogram showing HeLa cells stained with ab52459 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab52459, 1µg/1x106 cells) for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 (FITC) (2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
ICC/IF image of ab52459 stained HCT116 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 (ab52459, 5µg/ml) overnight at +4°C. 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.
References for Anti-Cytokeratin 18 antibody [C-04] (FITC) (ab52459)
This product has been referenced in:
Chauchereau A et al. Stemness markers characterize IGR-CaP1, a new cell line derived from primary epithelial prostate cancer. Exp Cell Res317:262-75 (2011).
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