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

Lanes 1 - 3: Merged signal (red and green). Green - ab16051 observed at 95 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab16051 was shown to specifically react with CTNNB1 (β-catenin) along with additional cross-reactive bands in wild-type HAP1 cells. No band was observed in knockout samples. Wild-type and CTNNB1 (β-catenin) knockout samples were subjected to SDS-PAGE. Ab16051 and ab9484 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 0.25 ug/ml and 1/20000 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.

ab16051 staining CTNNB1 (β-catenin) in HeLa cells. The cells were fixed with 100% methanol (5min) 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 ab16051 at 1μg/ml and ab7291 (staining Tubulin) at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an AlexaFluor®488 Goat anti-Rabbit secondary (ab150077) at 2 μg/ml (shown in green) and AlexaFluor®594 Goat anti-Mouse secondary (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.

IHC image of beta catenin staining in a formalin fixed, paraffin embedded normal human liver tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab16051, 0.5 µg/ml, 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.

 

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

IHC image of beta catenon staining in a formalin fixed, paraffin embedded human colon adenocarcinamo tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab16051, 0.5 µg/ml, 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.

 

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

ab16051 staining beta Catenin in rat brain tissue sections by Immunohistochemistry (IHC-P - formaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 2% BSA for 10 minutes at 21°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/1000 in TBS/BSA/azide) for 2 hours at 21°C. A Biotin-conjugated goat anti-rabbit IgG polyclonal (1/300) was used as the secondary antibody.

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ab16051 stained in MCF7cells. Cells were fixed with 100% methanol (5 min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab16051 at 5 µg/ml and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150120 (pseudo-colored red) and ab150081 (colored green) used at 1 ug/ml for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1hour at room temperature

All lanes : Anti-beta Catenin antibody (ab16051) at 0.25 µg/ml

Lane 1 : HeLa whole cell lysate
Lane 2 : HeLa whole cell lysate with Human beta Catenin peptide (ab16377) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Alexa Fluor Goat polyclonal to Rabbit IgG at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 94 kDa
Observed band size: 94 kDa
Additional bands at: 75 kDa. We are unsure as to the identity of these extra bands.

A second band of 75 kDa was also detected in Hela whole cell lysates and A431 lysates. This smaller band was of equal intensity to the 94 kDa band in the A431 lysates (data not shown).

Anti-beta Catenin antibody (ab16051) at 0.25 µg/ml + Recombinant Human beta Catenin protein (Tagged) (ab63175) at 0.01 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 94 kDa


Exposure time: 10 seconds

Standard Curve for Beta-Catenin (Analyte: beta Catenin protein (Tagged) (ab63175)); dilution range 1pg/ml to 1ug/ml using Capture Antibody Mouse monoclonal [BDI080] to beta Catenin (ab19448) at 1ug/ml and Detector Antibody Rabbit polyclonal to beta Catenin (ab16051) at 0.1ug/ml.

ab16051 staining beta Catenin in developing gut tissue sections from mouse by Immunohistochemistry (IHC-P - formaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 2% BSA for 2 minutes at 21°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/2000 in TBS/BSA/azide) for 2 hours at 21°C. A Biotin-conjugated goat anti-rabbit IgG polyclonal (1/300) was used as the secondary antibody.

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ab16051 staining β-catenin in SW480 cells treated with XAV939 (ab120897), by ICC/IF. Increase of ß-catenin cytoplasmic expression and decrease in nuclear expression correlates with increased concentration of XAV939, as described in literature.

The cells were incubated at 37°C for 6 hours in media containing different concentrations of ab120897 (XAV939) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab16051 (1 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

ab16051 staining human fibrosarcoma cells by ICC/IF.  Cells were PFA fixed and permeabilized in Triton X-100 prior to incubation with the primary antibody (at 10µg/ml) for 1 hour at 27°C.  A Texas Red® conjugated donkey anti-rabbit antibody was used as the secondary.

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