All lanes : Anti-Cytokeratin 7 antibody [EPR1619Y] - Cytoskeleton Marker (ab68459) 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
Observed band size: 50 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab68459 observed at 50 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab68459 was shown to specifically react with KRT7 (Cytokeratin 7) in wild-type A549 cells as signal was lost in KRT7 knockout cells. Wild-type and KRT7 knockout samples were subjected to SDS-PAGE. Ab68459 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-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue labelling Cytokeratin 7 with unpurified ab68459.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunocytochemistry/Immunofluorescence analysis of T47D cells labelling Cytokeratin 7 with purified ab68459 at a dilution of 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

Alexa Fluor^® 488 (ab185048) and Alexa Fluor^® 647 (ab192077) conjugated versions are available for this clone.

All lanes : Anti-Cytokeratin 7 antibody [EPR1619Y] - Cytoskeleton Marker (ab68459) at 1/10000 dilution (purified)

Lane 1 : HeLa whole cell lysate
Lane 2 : SK-OV-3 whole cell lysate
Lane 3 : T47D whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 51 kDa
Observed band size: 51 kDa



Blocking and dilution buffer: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human bladder carcinoma tissue labelling Cytokeratin 7 with purified ab68459 at a dilution of 1/1000. Heat mediated antigen retrieval was performed using EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Flow Cytometry analysis of HeLa cells labelling Cytokeratin 7 with purified ab68459 at a dilution of 1/20 (red). Cells were fixed with 80% methanol. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

Alexa Fluor^® 488 (ab185048) and Alexa Fluor^® 647 (ab192077) conjugated versions are available for this clone.

ab68459 (purified) at 1/20 immunoprecipitating Cytokeratin 7 in HeLa whole cell lysate.

Lane 1 (input): HeLa whole cell lysate (10µg)

Lane 2 (+): ab68459 + HeLa whole cell lysate.

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab68459 in HeLa whole cell lysate.

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

All lanes : Anti-Cytokeratin 7 antibody [EPR1619Y] - Cytoskeleton Marker (ab68459) at 1/10000 dilution (unpurified)

Lane 1 : HeLa cell lysate
Lane 2 : T47D cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 51 kDa
Observed band size: 51 kDa

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling Cytokerain 7 with unpurified ab68459 at a dilution of 1/100.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human endometrial carcinoma tissue labelling Cytokeratin 7 with unpurified ab68459.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human ovarian carcinoma tissue labelling Cytokeratin 7 with unpurified ab68459.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human urinary bladder transitional carcinoma tissue labelling Cytokeratin 7 with unpurified ab68459.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

ab68459 showing negative staining in human sarcoma tissue.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

ab68459 showing negative staining in human colonic adenocarcinoma tissue.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Overlay histogram showing HeLa cells stained with unpurified ab68459 (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 (unpurified ab68459, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

Equilibrium disassociation constant (K_D)
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