Anti-Claudin 4 antibody

• WB

Lab

Western blot - Anti-Claudin 4 antibody (AB53156)

False colour image of Western blot : Anti-Claudin 4 antibody staining at 1/500 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab53156 was shown to bind specifically to Claudin 4. A band was observed at 17 kDa in wild-type MCF7 cell lysates with no signal observed at this size in CLDN4 knockout cell line ab274946 (knockout cell lysate ab275004). To generate this image, wild-type and CLDN4 knockout MCF7 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Claudin 4 antibody (ab53156) at 1/500 dilution

Lane 1:

Wild-type MCF7 cell lysate at 20 µg

Lane 2:

Western blot - Human CLDN4 knockout MCF7 cell lysate (ab275004) at 20 µg

Lane 3:

PC-3 cell lysate at 20 µg

Lane 4:

U-2 OS cell lysate at 20 µg

Lane 5:

LNCaP cell lysate at 20 µg

Lane 6:

SW480 cell lysate at 20 µg

Lane 7:

HeLa cell lysate at 20 µg

Secondary

All lanes:

Western blot at 1/20000 dilution

Predicted band size: 22 kDa

Observed band size: 17 kDa

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• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Claudin 4 antibody (AB53156)

ICC/IF image of ab53156 staining Claudin 4 (green) in MCF7 (Human breast adenocarcinoma cell line) cells. The cells were fixed in 4% formaldehyde (10 minutes) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab53156, 5 μg/ml) overnight at +4°C. The secondary antibody was an Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1 hour. An Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1 hour. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43 μM.

• WB

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Western blot - Anti-Claudin 4 antibody (AB53156)

All lanes:

Western blot - Anti-Claudin 4 antibody (ab53156) at 1/500 dilution

Lane 1:

Untreated HeLa (Human epithelial cell line from cervix adenocarcinoma) cell extracts

Lane 2:

HeLa (Human epithelial cell line from cervix adenocarcinoma) cell extracts with immunizing peptide

Predicted band size: 22 kDa

Observed band size: 22 kDa

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