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.

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).

All lanes : Anti-beta Catenin antibody [EP690Y] (ab68183) at 1/1000 dilution (Purified)

Lane 1 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates
Lane 2 : C6 (Rat glial tumor glial cell) whole cell lysates

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: 86 kDa
Observed band size: 75,90 kDa why is the actual band size different from the predicted?

Blocking/Diluting buffer: 5% NFDM/TBST.

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

Anti-beta Catenin antibody [EP690Y] (ab68183) at 1/1000 dilution (Purified) + A431 (Human epidermoid carcinoma epithelial cell) whole cell lysates at 15 µg

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

Predicted band size: 86 kDa
Observed band size: 75,90 kDa why is the actual band size different from the predicted?

Blocking/Diluting buffer: 5% NFDM/TBST.

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

Immunocytochemistry/ Immunofluorescence analysis of parental HAP1 (Wildtype control Human chronic myelogenous leukemia near-haploid cell line) cells labeling beta Catenin with Purified ab68183 at 1/100 dilution (10 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.