Recombinant Anti-Cytokeratin 20 antibody [EPR1621(2)] (ab109111)

Anti-Cytokeratin 20 antibody [EPR1621(2)] (ab109111) at 1/10000 dilution (Purified) + SW480 (Human colorectal adenocarcinoma epithelial cell) whole cell lysate at 15 µg

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

Predicted band size: 48 kDa
Observed band size: 46-50 kDa why is the actual band size different from the predicted?

The molecule weight observed is consistent with what have been described in the literature (PMID: 8359595, 34001900).

Flow Cytometry analysis of HT-29 (Human colorectal adenocarcinoma epithelial cell) cells labelling Cytokeratin 20 with Purified ab109111 at 1:20 dilution (10 µg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1:2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunocytochemistry analysis of HT-29 (Human colorectal adenocarcinoma epithelial cell) cells labeling Cytokeratin 20 with Purified ab109111 at 1:50 dilution (3.1 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

All lanes : Anti-Cytokeratin 20 antibody [EPR1621(2)] (ab109111) at 1/10000 dilution

Lane 1 : HT-29 cell lysate
Lane 2 : Daudi cell lysate
Lane 3 : MCF7 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 48 kDa
Additional bands at: 45 kDa. We are unsure as to the identity of these extra bands.

This data was generated with the unpurified version of this antibody.

False colour image of Western blot: Anti-Cytokeratin 20 antibody [EPR1621(2)] staining at 1/10000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab109111 was shown to bind specifically to Cytokeratin 20. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

This data was generated with the unpurified version of this antibody.

Intracellular Flow Cytometry analysis of SW480 (human colorectal adenocarcinoma) cells labeling Cytokeratin 20 with unpurified ab109111 at 1/20 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) (ab150077) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) (ab172730) was used as the isotype control, Cell without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.