Recombinant Anti-beta Catenin antibody [EP690Y] (ab68183)

Blocking and diluting buffer and concentration: 5% NFDM/TBST.

ab181602 was used as a GAPDH loading control.

Raw264.7 expresses low level of beta Catenin and stimulation is required to allow detection of the beta Catenin protein in this cell line, as described in PMID: 22983902 and PMID: 29137395.

False colour image of Western blot: Anti-beta Catenin antibody [EP690Y] 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, ab68183 was shown to bind specifically to beta Catenin. A band was observed at 85 kDa in wild-type HepG2 cell lysates with no signal observed at this size in CTNNB1 knockout cell line. To generate this image, wild-type and CTNNB1 knockout HepG2 cell lysates were analysed. 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.

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: CTNNB1 (β-Catenin) knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: A431 whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab68183 observed at 85 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab68183 was shown to specifically react with CTNNB1 (β-Catenin) in wild-type HAP1 cells along with additional cross reactive bands. No band was observed when knockout samples were used. Wild-type and CTNNB1 (β-Catenin) knockout samples were subjected to SDS-PAGE. ab68183 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/500 dilution and 1/10000 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.

This image was generated using un-purified format of the antibody.

ab68183 staining β-catenin in CTNNB1 (β-Catenin) wild-type HAP1 cells (top panel) and CTNNB1 (β-Catenin) knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), 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 ab68183 at 1/250 dilution and ab195889 at 1/250 dilution (shown in pseudocolour red) 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). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This image was generated using un-purified format of the antibody.

Intracellular Flow Cytometry analysis of A431 (Human epidermoid carcinoma epithelial cell) cells labeling beta Catenin with Purified ab68183 at 1/20 dilution (5 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). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Blocking/Diluting buffer: 5% NFDM/TBST.

Full-length beta catenin: 90kDa ; C-terminal cleavage fragment: 75kDa (PMID: 15492240).

Blocking/Diluting buffer: 5% NFDM/TBST.

Full-length beta catenin: 90kDa ; C-terminal cleavage fragment: 75kDa (PMID: 15492240).