All lanes : Anti-Cytokeratin 14 antibody [EPR17336] (ab197893) at 1/50000 dilution

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

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 66 kDa
Observed band size: 52 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab197893 observed at 52 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab197893 was shown to react with KRT14 in A431 wild-type cells in Western blot. Loss of signal was observed when KRT14 knockout sample was used. A431 wild-type 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 ab197893 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 50000 dilution and a 1 in 20000 dilution respectively. Blots were developed 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.

Immunohistochemical analysis of paraffin-embedded Humansquamous cell carcinoma of cervix tissue labeling Cytokeratin 14 using ab197893 at 1/500 dilution. A Goat Anti-Rabbit IgG H&L (HRP) (ab97051) was used as secondary at 1/500 dilution. Counterstain: Hematoxylin.
Inset image: negative control obtained using PBS instead of ab197893 and secondary antibody only.
Note: Cytoplasm staining on human squamous cell carcinoma of cervix tissue was observed.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Mouse skin tissue labeling Cytokeratin 14 using ab197893 at 1/500 dilution. A Goat Anti-Rabbit IgG H&L (HRP) (ab97051) was used as secondary at 1/500 dilution. Counterstain: Hematoxylin.
Inset image: negative control obtained using PBS instead of ab197893 and secondary antibody only.
Note: Cytoplasm staining on mouse skin tissue was observed.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

All lanes : Anti-Cytokeratin 14 antibody [EPR17336] (ab197893) at 1/50000 dilution

Lane 1 : Mouse skin lysate at 20 µg
Lane 2 : Rat skin lysate at 20 µg
Lane 3 : Rat brain lysate at 10 µg
Lane 4 : Rat heart lysate at 10 µg
Lane 5 : Rat kidney lysate at 10 µg
Lane 6 : Rat spleen lysate at 10 µg
Lane 7 : C6(Rat glial tumor cells) whole cell lysate at 10 µg
Lane 8 : Raw264.7(Mouse macrophage cells transformed with Abelson murine leukemia virus ) whole cell lysate at 10 µg
Lane 9 : PC-12(Rat adrenal gland pheochromocytoma) whole cell lysate at 10 µg
Lane 10 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate at 10 µg

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 66 kDa
Observed band size: 53 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

5% NFDM/TBST: Blocking and diluting buffer.

The expression of Cytokeratin 14 is basal cells of epidermis and other stratified epithelia. Lanes 3-10 represent Cytokeratin 14 negative tissues and cell lines.

Anti-Cytokeratin 14 antibody [EPR17336] (ab197893) at 1/50000 dilution + human fetal skin lysate at 10 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 66 kDa
Observed band size: 53 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

5% NFDM/TBST: Blocking and diluting buffer.