All lanes : Anti-beta Catenin antibody [IGX4794R-3] (ab223075) at 1 µg/ml

Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : CTNNB1 knockout HCT116 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 85 kDa
Observed band size: 95 kDa why is the actual band size different from the predicted?

Lanes 1 - 2: Merged signal (red and green). Green - ab223075 observed at 95 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab223075 was shown to react with Anti-beta Catenin in HCT 116 wild-type cells in western blot with loss of signal observed in CTNNB1 knockout cell line ab273712 (CTNNB1 knockout cell lysate ab275247). HCT 116 wild-type and CTNNB1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluoroscent western blot (TBS-based) blocking solution 50% (v/v) in TBS-T (0.1% Tween^®) before incubation with ab223075 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at 1 µg/ml and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 hour at room temperature before imaging.

ab223075 staining β-Catenin in 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 ab223075 at 0.05μg/ml and ab195889 at 1/250 dilution (shown in 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).

IHC image of CTNNB1 staining in a section of formalin-fixed paraffin-embedded human colon (normal)*, performed on a Leica Bond^TM. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins, before blocking of endogenous biotin using ab64212. The section was then incubated with ab223075, 0.25ug/ml, for 15 mins at room temperature and detected using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

All lanes : Anti-beta Catenin antibody [IGX4794R-3] (ab223075) at 1 µg/ml

Lane 1 : A549 (Human lung adenocarcinoma epithelial cell line) Whole Cell Lysate
Lane 2 : Caco 2 (Human colonic carcinoma cell line) Whole Cell Lysate
Lane 3 : HUES7 (Human embryonic stem cell line) Whole Cell Lysate
Lane 4 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lane 5 : mES (Mouse embryonic stem cell) Whole Cell Lysate
Lane 6 : E14Tg2a (Mouse embryonic stem cell line) Whole Cell Lysate
Lane 7 : Colon (Human) Tissue Lysate - adult normal tissue
Lane 8 : Wild type HAP1 whole cell lysate
Lane 9 : Beta Catenin knockout HAP1 whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/50000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 85 kDa
Observed band size: 92 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before being incubated with ab223075 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.

Lane 1: Wild type HAP1 whole cell lysate (10 µg)
Lane 2: CTNNB1 (β-Catenin) knockout HAP1 whole cell lysate (10 µg)
Lane 3: A431 whole cell lysate (10 µg)
Lane 4: Caco-2 whole cell lysate (10 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab223075 observed at 95 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab223075 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. ab223075 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 µg/ml 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.

ab223075 staining CTNNB1 in MCF-7 cells. The cells were fixed with 100% methanol (5 min), 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 ab223075 at a 0.05µg/ml concentration, then detected with an Alexa Fluor^® 488 goat anti-rabbit secondary antibody (ab150081) at a 1/1000 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue), and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at a 1/250 dilution (shown in red).

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

This product also gave a positive signal in 4% formaldehyde (10 min) fixed MCF-7 cells under the same testing conditions.

IHC image of CTNNB1 staining in a section of formalin-fixed paraffin-embedded human colon adenocarcinoma*, performed on a Leica BOND^TM. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins.  The section was then incubated with ab223075, 0.1ug/ml dilution, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre