Recombinant Alexa Fluor® 647 Anti-beta Catenin antibody [E247] (ab194119)

Overlay histogram showing HAP1 wildtype (green line) and HAP1-CNNB1 knockout cells (red line) stained with ab194119. The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab194119, 0.01µg/ml) for 30 min at 22°C.

A mouse IgG1 isotype control antibody  (ab199093) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-CNNB1 knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5,000 events were collected using a 40 mW Red laser (640nm) and 670/14 bandpass filter.

This antibody can also be used in HAP1 cells fixed with 4% formaldehyde (10 min) permeabilized with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

ab194119 staining β-catenin in wild-type HAP1 cells (top panel) and 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 ab194119 at 1/500 dilution (shown in red) and ab195887 at 1/250 dilution (shown in green) overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.

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

ab194119 staining beta Catenin in Caco-2 cells. The cells were fixed with 4% formaldehyde (10min), 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 overnight at +4°C with ab194119 at 1/100 dilution (shown in red) and ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor® 488), at 2µg/ml (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab194119 staining beta Catenin in SW480 cells. The cells were fixed with 4% formaldehyde (10 min), permeabilised in 0.1% Triton X-100 for 5 minutes and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab194119 at a working dilution of 1 in 50 (shown in red) and ab195887, Mouse monoclonal [DM1A] to alpha Tubulin (Alexa Fluor^® 488, shown in green) at 2µg/ml overnight at +4°C. Nuclear DNA was labelled in blue with DAPI.

This product gave a positive signal in 100% methanol (5 min) fixed SW480 cells under the same testing conditions.

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