ab52625 staining Cytokeratin 19 in wild-type MCF7 cells (top panel) and KRT19 knockout MCF7 cells (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min), 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 ab52625 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).

Immunohistochemical staining of paraffin-embedded human skin with purified ab52625 at a dilution of 1/400. A pre-diluted HRP polymer for rabbit/mouse IgG was used as the secondary antibody and the sample was counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Immunocytochemistry/Immunofluorescence analysis of HepG2 (human liver hepatocellular carcinoma cell line) cells labelling Cytokeratin 19 (green) with purified ab52625 at 1/500. Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. Cells were counter-stained with ab7291, anti-Tubulin (mouse mAb) at 1/1000 followed by ab150120 Alexa Fluor^® 594 goat anti-mouse secondary (1/1000). Nuclei were counterstained with DAPI (blue).

For negative control 1, rabbit primary antibody and anti-mouse secondary antibody (ab150120) were used. For negative control 2, ab7291 (mouse primary antibody) was used followed by anti-rabbit secondary antibody (ab150077).

Alexa Fluor^® 488 (ab192643) and Alexa Fluor^® 647 (ab192980) conjugated versions are available for this clone.

All lanes : Anti-Cytokeratin 19 antibody [EP1580Y] - Cytoskeleton Marker (ab52625) at 1/45000 dilution (purified)

Lane 1 : HepG2 (liver hepatocellular carcinoma cell line) cell lysate
Lane 2 : NIH/3T3 (mouse embyro fibroblast cell line) cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit (H+L) at 1/1000 dilution

Predicted band size: 44 kDa
Observed band size: 40 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

ab52625 staining Cytokeratin 19 in the human cell line MCF-7 (human breast carcinoma) by flow cytometry. Cells were fixed with 4% paraformaldehyde, permeabilized with 90% methanol and the sample was incubated with the primary antibody at a dilution of 1/80. A goat anti rabbit IgG (Alexa Fluor^® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control: Rabbit monoclonal IgG (Black)

Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue).

Alexa Fluor^® 488 (ab192643) and Alexa Fluor^® 647 (ab192980) conjugated versions are available for this clone.

Unpurified ab52625 showing positive staining in Breast carcinoma tissue.

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

Immunofluorescent staining of MCF-7 cells (fixed in 4% PFA, permeabilized with 0.1% Triton X 100) using purified ab52625 at a dilution of 1/200. An Alexa Fluor^® 488 goat anti-rabbit antibody was used as the secondary at a dilution of 1/200 and the cells were counter stained with DAPI.

Overlay histogram showing HeLa (human epithelial cell line from cervix adenocarcinoma) cells stained with unpurified ab52625 (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 (ab52625, 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. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

Unpurified ab52625 showing positive staining in Endometrial carcinoma tissue.

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

Unpurified ab52625 showing negative staining in Glioma tissue.

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

Unpurified ab52625 showing positive staining in Gastric adenocarcinoma tissue.

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

Equilibrium disassociation constant (K_D)
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