Anti-beta Catenin antibody [EP690Y]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-beta Catenin antibody [EP690Y] (AB68183)

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).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-beta Catenin antibody [EP690Y] (AB68183)

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-beta Catenin antibody [EP690Y] (AB68183)

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.

• WB

Lab

Western blot - Anti-beta Catenin antibody [EP690Y] (AB68183)

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.

All lanes:

Western blot - Anti-beta Catenin antibody [EP690Y] (ab68183)

Predicted band size: 85 kDa

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• WB

Lab

Western blot - 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.

All lanes:

Western blot - Anti-beta Catenin antibody [EP690Y] (ab68183) at 1/1000 dilution

Lane 1:

RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 2:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 3:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 86 kDa

Observed band size: 90 kDa

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Exposure time: 180s

• WB

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Western blot - Anti-beta Catenin antibody [EP690Y] (AB68183)

Blocking/Diluting buffer : 5% NFDM/TBST.

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

All lanes:

Western blot - Anti-beta Catenin antibody [EP690Y] (ab68183) at 1/1000 dilution

All lanes:

A431 (Human epidermoid carcinoma epithelial cell) whole cell lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 85 kDa

Observed band size: 75 kDa,90 kDa

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• WB

Lab

Western blot - Anti-beta Catenin antibody [EP690Y] (AB68183)

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.

All lanes:

Western blot - Anti-beta Catenin antibody [EP690Y] (ab68183) at 1/500 dilution

Lane 1:

Wild-type HepG2 cell lysate at 20 µg

Lane 2:

CTNNB1 knockout HepG2 cell lysate at 20 µg

Lane 2:

Western blot - Human CTNNB1 (beta Catenin) knockout Hep G2 cell line (<a href='/en-us/products/cell-lines/human-ctnnb1-beta-catenin-knockout-hep-g2-cell-line-ab277911'>ab277911</a>)

Lane 3:

HeLa cell lysate at 20 µg

Lane 4:

A431 cell lysate at 20 µg

Predicted band size: 85 kDa

Observed band size: 85 kDa

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• WB

Unknown

Western blot - Anti-beta Catenin antibody [EP690Y] (AB68183)

Blocking/Diluting buffer : 5% NFDM/TBST.

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

All lanes:

Western blot - Anti-beta Catenin antibody [EP690Y] (ab68183) at 1/1000 dilution

Lane 1:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates at 20 µg

Lane 2:

C6 (Rat glial tumor glial cell) whole cell lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 85 kDa

Observed band size: 75 kDa,90 kDa

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