Anti-beta Catenin antibody [IGX4794R-3]

9 product images

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  Western blot - Anti-beta Catenin antibody [IGX4794R-3] (ab223075)

  Beta Catenin Western blot staining using rabbit Anti-beta Catenin antibody

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

  ab223075 was shown to react with Anti-beta Catenin in wild-type HCT 116 cells in western blot with loss of signal observed in CTNNB1 knockout cell line Human CTNNB1 knockout HCT116 cell line ab273712 (CTNNB1 knockout cell lysate Human CTNNB1 (beta Catenin) knockout HCT116 cell lysate ab275247). Wild-type and CTNNB1 knockout HCT 116 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 Anti-GAPDH antibody [6C5] - Loading Control 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

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

  Lane 1: Wild-type HCT116 cell lysate at 20 µg

  Lane 2: Western blot - Human CTNNB1 (beta Catenin) knockout HCT116 cell lysate (Human CTNNB1 (beta Catenin) knockout HCT116 cell lysate ab275247)

  Lane 2: Western blot - Human CTNNB1 knockout HCT116 cell line (Human CTNNB1 knockout HCT116 cell line ab273712)

  Lane 2: CTNNB1 knockout HCT116 cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 85 kDa

  Observed band size: 95 kDa

• 

  Immunocytochemistry/ Immunofluorescence - Anti-beta Catenin antibody [IGX4794R-3] (ab223075)

  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 Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed 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).

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta Catenin antibody [IGX4794R-3] (ab223075)

  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 Avidin/Biotin Blocking Kit 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

• 

  Western blot - Anti-beta Catenin antibody [IGX4794R-3] (ab223075)

  Beta Catenin Western blot staining using rabbit Anti-beta Catenin antibody

  False colour image of Western blot: Anti-beta Catenin antibody [IGX4794R-3] staining at 1 ug/ml, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab223075 was shown to bind specifically to beta Catenin. A band was observed at 95 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in CTNNB1 knockout cell line Human CTNNB1 knockout HCT116 cell line ab273712 (knockout cell lysate Human CTNNB1 (beta Catenin) knockout HCT116 cell lysate ab275247). The band observed in the knockout lysate lane below 95 kDa is likely to represent a truncated form of beta Catenin. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and CTNNB1 knockout HCT 116 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.

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

  Lane 1: Wild-type HCT 116 cell lysate at 20 µg

  Lane 2: Western blot - Human CTNNB1 (beta Catenin) knockout HCT116 cell lysate (Human CTNNB1 (beta Catenin) knockout HCT116 cell lysate ab275247)

  Lane 2: Western blot - Human CTNNB1 knockout HCT116 cell line (Human CTNNB1 knockout HCT116 cell line ab273712)

  Lane 2: CTNNB1 knockout HCT 116 cell lysate at 20 µg

  Secondary

  Lanes 1 - 2: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution

  Lanes 1 - 2: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 85 kDa

  Observed band size: 95 kDa

• 

  Western blot - Anti-beta Catenin antibody [IGX4794R-3] (ab223075)

  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 ECL Substrate Kit (High Sensitivity) ab133406.

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

  Lane 1: A549 (Human lung adenocarcinoma epithelial cell line) Whole Cell Lysate at 10 µg

  Lane 2: Caco 2 (Human colonic carcinoma cell line) Whole Cell Lysate at 10 µg

  Lane 3: HUES7 (Human embryonic stem cell line) Whole Cell Lysate at 10 µg

  Lane 4: NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate at 10 µg

  Lane 5: mES (Mouse embryonic stem cell) Whole Cell Lysate at 10 µg

  Lane 6: E14Tg2a (Mouse embryonic stem cell line) Whole Cell Lysate at 10 µg

  Lane 7: Colon (Human) Tissue Lysate - adult normal tissue at 10 µg

  Lane 8: Wild type HAP1 whole cell lysate at 10 µg

  Lane 9: Beta Catenin knockout HAP1 whole cell lysate at 10 µg

  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

  Exposure time: 3min

• 

  Western blot - Anti-beta Catenin antibody [IGX4794R-3] (ab223075)

  False colour image of Western blot: Anti-beta Catenin antibody [IGX4794R-3] staining at 1 ug/ml, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab223075 was shown to bind specifically to beta Catenin. A band was observed at 95 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in CTNNB1 CRISPR-Cas9 edited cell line Human CTNNB1 knockout HCT116 cell line ab273712 (CRISPR-Cas9 edited cell lysate Human CTNNB1 (beta Catenin) knockout HCT116 cell lysate ab275247). The band observed in the CRISPR-Cas9 edited lysate lane below 95 kDa is likely to represent a truncated form of beta Catenin. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and CTNNB1 CRISPR-Cas9 edited HCT 116 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.

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

  Lane 1: Wild-type HCT 116 cell lysate at 20 µg

  Lane 2: CTNNB1 CRISPR-Cas9 edited HCT 116 cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 85 kDa

  Observed band size: 95 kDa

• 

  Western blot - Anti-beta Catenin antibody [IGX4794R-3] (ab223075)

  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, Anti-GAPDH antibody [mAbcam 9484] - 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 Anti-GAPDH antibody [mAbcam 9484] - Loading Control 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 Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-beta Catenin antibody [IGX4794R-3] (ab223075)

  Predicted band size: 85 kDa

• 

  Immunocytochemistry/ Immunofluorescence - Anti-beta Catenin antibody [IGX4794R-3] (ab223075)

  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 (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at a 1/1000 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue), and Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta Catenin antibody [IGX4794R-3] (ab223075)

  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