All lanes : HRP Anti-Cytokeratin 7 antibody [EPR17078] (ab209945) at 1/5000 dilution

Lane 1 : Wild-type A549 whole cell lysate
Lane 2 : KRT7 knockout A549 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : MCF7 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 51 kDa

ab209945 was shown to specifically react with KRT7 in wild-type A549 cells as signal was lost in KRT7 knockout cells. Wild-type and KRT7 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab209945 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/5000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibody at 1/20000 dilution for 1 hour at room temperature. The loading control was imaged using the Licor Odyssey CLx prior to blots being developed with ECL technique.

IHC image of Cytokeratin 7 staining in a section of formalin-fixed paraffin-embedded normal human breast*, performed on a Leica BOND™. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab209945, 1/100 dilution, for 15 mins at room temperature. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.

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.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

All lanes : HRP Anti-Cytokeratin 7 antibody [EPR17078] (ab209945) at 1/5000 dilution

Lane 1 : Lung (Mouse) Tissue Lysate
Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 51 kDa
Observed band size: 53 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab209945 overnight at 4°C. Antibody binding was visualised using ECL development solution ab133406.