重组Anti-Cytokeratin 14抗体[EP1612Y] (ab51054)

Lanes 1 - 4: Merged signal (red and green). Green - ab51054 observed at 49 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab51054 was shown to react with Cytokeratin 14 in wild-type A431 cells in western blot. Loss of signal was observed when KRT14 knockout sample was used. Wild-type A431 and KRT14 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab51054 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Purified ab51054 at 1/20 dilution (0.5µg) immunoprecipitating Cytokeratin 14 in A431 whole cell lysate.
Lane 1 (input): A431 (Human epidermoid carcinoma epithelial cell) whole cell lysate 10µg
Lane 2 (+): ab51054 + A431 whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab51054 in A431 whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 48 kDa

Overlay histogram showing A431 (Human epidermoid carcinoma cell line) cells stained with ab51054 (red line). 

The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton 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 (ab51054, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. 

This antibody gave a positive signal in A431 cells fixed with 4% paraformaldehyde/permeabilized in 0.1% PBS-Triton used under the same conditions. 

ab51054 staining Cytokeratin 14 in human skin tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections).

Tissue was fixed in paraformaldehyde and a heat mediated antigen retrieval step was performed using citrate buffer, pH 6.0. Samples were then permeabilized using 0.1% saponin/PBS, blocked with 4% BSA for 30 minutes at 25°C and then incubated with ab51054 at a 1/200 dilution for 16 hours at 4°C. The secondary used was a Texas Red conjugated goat anti-rabbit polyclonal used at a 1/100 dilution.

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ICC/IF image of ab51504 stained A431 (Human epidermoid carcinoma cell line) cells.

The cells were fixed in 100% methanol (5 min) and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab51504, 1/100 dilution) 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 1.43 µM.