Immunohistochemical analysis of paraffin-embedded human squamous lung carcinoma tissue sections labeling Cytokeratin 14 with purified ab51054 at 1/100 dilution. ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Sections were counterstained with Hematoxylin.
Antigen retrieval was heat mediated antigen retrieval using citrate buffer, pH 6.0).

ab51054 staining KRT14 in wild-type A431 cells (top panel) and KRT14 knockout A431 cells (bottom panel). The cells were fixed with 100% methanol (5 min) then permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab51054 at 1/100 dilution and ab7291 (Mouse monoclonal to alpha Tubulin) at 1/1000 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor^® 594) (ab150120) at 2 μg/ml (shown in red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

All lanes : Anti-Cytokeratin 14 antibody [EP1612Y] (ab51054) at 1/10000 dilution

Lane 1 : Wild-type A431 cell lysate
Lane 2 : KRT14 knockout A431 cell lysate
Lane 3 : Human skin cell lysate
Lane 4 : MCF7 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 52 kDa
Observed band size: 49 kDa why is the actual band size different from the predicted?

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.

Anti-Cytokeratin 14 antibody [EP1612Y] (ab51054) at 1/20000 dilution + A431 (Human epidermoid carcinoma cell line) cell lysate at 10 µg

Secondary
Goat anti-Rabbit-HRP at 1/2000 dilution

Predicted band size: 52 kDa
Observed band size: 48 kDa why is the actual band size different from the predicted?