Anti-beta Catenin antibody [E247] - ChIP Grade

39 product images

• 

  Image from Jin et al PLoS One. 2015 Aug 7;10(8):e0133770. doi: 10.1371/journal.pone.0133770. eCollection 2015. Fig 2.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Different expression level of beta Catenin in HCTs (hepatocellular carcinoma tissues) and PLTs (para-cancerous liver tissues).

  The HCTs, PLTs were paraffin-embedded and cut into sections with 5 μm-thickness for hematoxylin-eosin and immunohistochemistry (IHC) analysis. ab32572 was used at a dilution of 1:400. The second antibody was a biotinylated IgG to incubate 40 minutes at 37°C. Finally, the tissue slices were visualized by the 3, 3-diaminobenzidine solution and counterstained with hematoxylin. Substitution of the primary antibody with phosphate-buffered saline was served as a control for IHC.

  The beta Catenin with negative, weak, moderate and strong staining activity was respectively detected in HCTs (E-H) and PLTs (M-P). Section E shown above, for full image please see original paper.

• 

  Western blot - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  False colour image of Western blot: Anti-beta Catenin antibody [E247] - ChIP Grade staining at 1/5000 dilution, 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, ab32572 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 5 % 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 [E247] - ChIP Grade (ab32572) at 1/5000 dilution

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

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

  Performed under reducing conditions.

  Predicted band size: 85 kDa

  Observed band size: 95 kDa

• 

  Immunoprecipitation - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  beta Catenin was immunoprecipitated from 0.35 mg mouse brain lysate with ab32572 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab32572 1/1000 dilution (1.962 μg/ml). VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) was used as the secondary antibody at 1/1000 dilution.
  

  Lane 1: Mouse brain tissue lysate 10 μg

  Lane 2: Mouse brain tissue lysate

  Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab32572 in mouse brain lysate.

  Blocking and dilution buffer and concentration: 5% NFDM/TBST.

  Exposure time: 1 seconds

  All lanes: Immunoprecipitation - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Predicted band size: 85 kDa

• 

  Western blot - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  False colour image of Western blot: Anti-beta Catenin antibody [E247] - ChIP Grade staining at 1/5000 dilution, 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, ab32572 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 (Human CTNNB1 (beta Catenin) knockout Hep G2 cell line ab277911). 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 (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 [E247] - ChIP Grade (ab32572) at 1/5000 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 (Human CTNNB1 (beta Catenin) knockout Hep G2 cell line ab277911)

  Lane 3: HeLa cell lysate at 20 µg

  Lane 4: A431 cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 85 kDa

  Observed band size: 85 kDa

• 

  Western blot - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Lanes 1 - 4: Merged signal (red and green). Green - ab32572 observed at 90 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245, observed at 37 kDa.
  

  ab32572 was shown to specifically react with CTNNB1 (β-catenin) in wild type HAP1 cells. No band was observed when CTNNB1 (β-catenin) knockout samples were used. Wild-type and CTNNB1 (β-catenin) knockout samples were subjected to SDS-PAGE. ab32572 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at a 1/5000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (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 [E247] - ChIP Grade (ab32572) at 1/5000 dilution

  Lane 1: Wild-type HAP1 whole cell lysate at 20 µg

  Lane 2: CTNNB1 (β-catenin) knockout HAP1 whole cell lysate at 20 µg

  Lane 3: HeLa whole cell lysate at 20 µg

  Lane 4: A431 whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/5000 dilution

  Predicted band size: 85 kDa

• 

  Western blot - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Lanes 1- 2: Merged signal (red and green). Green - ab32572 observed at 86 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) observed at 37 kDa.
  

  ab32572 was shown to react with beta Catenin in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line Human CTNNB1 (beta Catenin) knockout HeLa cell line ab255352 (knockout cell lysate Human CTNNB1 (beta Catenin) knockout HeLa cell lysate ab263756) was used. Wild-type HeLa and CTNNB1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab32572 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) overnight at 4°C at a 1 in 500 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (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 [E247] - ChIP Grade (ab32572) at 1/500 dilution

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

  Lane 2: CTNNB1 knockout HeLa cell lysate at 20 µg

  Lane 2: Western blot - Human CTNNB1 (beta Catenin) knockout HeLa cell line (Human CTNNB1 (beta Catenin) knockout HeLa cell line ab255352)

  Performed under reducing conditions.

  Predicted band size: 85 kDa

  Observed band size: 86 kDa

• 

  Immunocytochemistry/ Immunofluorescence - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  ab32572 staining in CTNNB1 (beta Catenin) wild-type HAP1 cells (top panel) and in 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 ab32572 at 1/250 dilution and Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) secondary antibody at 2 μg/ml (shown in green). Nuclear DNA was labeled in blue with DAPI.
  

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

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Immunohistochemical analysis of paraffin-embedded rat liver pancreas labeling beta Catenin with ab32572 at 1/1000 dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Membranous staining on rat pancreas. The section was incubated with ab32572 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins
  

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).

• 

  Immunoprecipitation - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  beta Catenin was immunoprecipitated from 0.35 mg A431 (Human epidermoid carcinoma epithelial cell) whole cell lysate with ab32572 at 1/50 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab32572 1/500 dilution (2 μg/ml). VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) was used as the secondary antibody at 1/1000 dilution.
  

  Lane 1: A431 (Human epidermoid carcinoma epithelial cell) whole cell lysate 10μg

  Lane 2: ab32572 IP in A431 whole cell lysate

  Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab32572 in A431 whole cell lysate.

  Blocking and dilution buffer and concentration: 5% NFDM/TBST.

  Exposure time: 3 seconds

  Lane 1: Immunoprecipitation - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572) at 1/500 dilution

  Lanes 2 - 3: Immunoprecipitation - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Lane 1: A431 (Human epidermoid carcinoma epithelial cell) whole cell lysate at 10 µg

  Lane 2: ab32572 IP in A431 whole cell lysate

  Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab32572 in A431 whole cell lysate

  Secondary

  All lanes: Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/1000 dilution

  Predicted band size: 85 kDa

  Observed band size: 90 kDa

• 

  Western blot - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

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

  False colour image of Western blot: Anti-beta Catenin antibody [E247] - ChIP Grade staining at 1/5000 dilution, 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, ab32572 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 5 % 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 [E247] - ChIP Grade (ab32572) at 1/5000 dilution

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

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

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

  Performed under reducing conditions.

  Predicted band size: 85 kDa

  Observed band size: 95 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Immunohistochemical analysis of paraffin-embedded rat liver tissue labeling beta Catenin with ab32572 at 1/1000 dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Membranous staining on rat liver. The section was incubated with ab32572 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins
  

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Immunohistochemical analysis of paraffin-embedded mouse pancreas tissue labeling beta Catenin with ab32572 at 1/1000 dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Membranous staining on mouse pancreas. The section was incubated with ab32572 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins
  

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).

• 

  Western blot - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Different batches of ab32572 were tested on A431 (Human epidermoid carcinoma epithelial cell) lysate at 2.0 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 92 kDa.

  All lanes: Western blot - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Predicted band size: 85 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Immunohistochemical analysis of paraffin-embedded mouse liver tissue labeling beta Catenin with ab32572 at 1/1000 dilution, followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Membranous staining on mouse liver. The section was incubated with ab32572 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins
  

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Immunohistochemical analysis of paraffin-embedded Human breast carcinoma tissue labeling beta Catenin with ab32572, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Membranous staining on human breast carcinoma.The section was incubated with Anti-DLL3 antibody [EPR22592-18] ab229902 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0).
  

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

• 

  Western blot - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Blocking/Diluting buffer and concentration: 5% NFDM/TBST.
  

  Exposure time: 180 seconds

  All lanes: Western blot - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572) at 1/10000 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) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Predicted band size: 85 kDa

  Observed band size: 92 kDa

• 

  Immunocytochemistry/ Immunofluorescence - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  ab32572 staining beta Catenin in SW480 (Human colorectal adenocarcinoma cell line) cells treated with BIO (BIO, GSK3 inhibitor; cell-permeable ab120891), by ICC/IF. Increase of beta Catenin expression correlates with increased concentration of BIO, as described in literature.
  The cells were incubated at 37°C for 48h in media containing different concentrations of BIO, GSK3 inhibitor; cell-permeable ab120891 (BIO) 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 ab32572 (1/200) dilution was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed (Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed ab96899) secondary antibody at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

• 

  Immunocytochemistry/ Immunofluorescence - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  ab32572 staining beta Catenin in SK-N-SH (Human neuroblastoma cell line) cells treated with olanzapine (Olanzapine, 5-HT, dopamine, histamine and muscarinic antagonist ab120736), by ICC/IF.
  

  Increase in expression of beta Catenin correlates with increased concentration of olanzapine, as described in literature.
  The cells were incubated at 37°C for 24h in media containing different concentrations of Olanzapine, 5-HT, dopamine, histamine and muscarinic antagonist ab120736 (olanzapine) 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 ab32572 (1/200 dilution) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed (Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed ab96899) secondary antibody at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

• 

  Olsen et al PLoS One. 2014 Dec 23;9(12):e115496. doi: 10.1371/journal.pone.0115496. eCollection 2014. Fig 3. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

  Western blot - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  WB analysis of total cell extracts from WT and gene disrupted cells using ab32572 at a 1/5000 dilution together with anti actin antibody. The position and full length β-catenin, truncated β-catenin and actin bands are indicated. For wild type cells 5 μg of TP and for the gene disrupted clones 30 μg of TP was applied for each lane.
  

  Cells were lysed in RIPA buffer containing protease inhibitor and phosphatase inhibitor tablets. Cell lysates were cleared by centrifugation and protein concentration 5–30 μg of total protein in SDS sample buffer was loaded per lane and separated.

  Secondary antibody was a donkey anti-rabbit IgG-HRP used at a 1∶5000 dilution.

  All lanes: Western blot - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Developed using the ECL technique.

  Predicted band size: 85 kDa

• 

  This image is courtesy of an anonymous customer review

  Western blot - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Western blot image of ab32572 staining whole cell lysate of U-2 OS (Human bone osteosarcoma epithelial cell line) cells. The gel was blocked with 5% milk for 1 hour at 21°C. The primary antibody was diluted 1/5000 and incubated for 12 hours at 4°C. An HRP conjugated swine anti-rabbit antibody was used as the secondary.

  All lanes: Western blot - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572) at 1/5000 dilution

  All lanes: U-2 OS (Human bone osteosarcoma epithelial cell line) whole cell lysate

  Performed under reducing conditions.

  Predicted band size: 85 kDa

  Observed band size: 90 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Immunohistochemical analysis of human cervical carcinoma tissue staining beta Catenin with ab32572 at 1/500 dilution. Heat mediated antigen retrieval was perfomed with citrate buffer (pH 6)

• 

  Western blot - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Brain (Mouse).
  

  Blocking with 5% milk. The blocking time os 1 hour at 22°C.

  Detected by ECL. Exposure time: 5 seconds.

  All lanes: Western blot - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572) at 1/1000 dilution

  All lanes: Brain (mouse) whole tissue lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 85 kDa

• 

  Western blot - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Rat Pericyte cells.
  

  Blocking and dilution buffer and concentration: 5% Milk. Blocking time 1 hour and temperature at 22°C

  Exposure time: 10 seconds

  All lanes: Western blot - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572) at 1/1000 dilution

  All lanes: Rat Pericytes whole cell lysate

  Performed under reducing conditions.

  Predicted band size: 85 kDa

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Immunohistochemical analysis of human lung adenocarcinoma tissue staining beta Catenin with ab32572 at 1/500 dilution. Heat mediated antigen retrieval was perfomed with citrate buffer (pH 6).

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Immunohistochemical analysis of human papillary carcinoma tissue staining beta Catenin with ab32572 at 1/500 dilution. Heat mediated antigen retrieval was perfomed with citrate buffer (pH 6).

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  ab32572 showing positive staining in human kidney carcinoma tissue.
  

  Immunohistochemical analysis of human kidney carcinoma tissue staining beta Catenin with ab32572 at 1/500 dilution. Heat mediated antigen retrieval was perfomed with citrate buffer (pH 6).

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Tissue Microarrays stained for Anti-beta Catenin antibody [E247] - ChIP Grade using ab32572 in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negaive (cross mark) staining per sample type tested. The section was incubated with ab32572 for 30 mins at room temperature followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). The immunostaining was performed on a Leica Biosystems BOND® RX instrument.
  
  Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

• 

  Multiplex immunohistochemistry - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Beta Catenin Multiplex immunohistochemistry staining of Mouse liver using rabbit Anti-beta Catenin antibody

  Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded mouse liver tissue staining ALDH1L1 with Anti-ALDH1L1 antibody [EPR25443-103] - Astrocyte Marker ab307696 at a 1/5000 dilution, Anti-HIRA/HIR antibody [EPR25299-11] ab302928 anti-HIRA/HIR used at 1/100 dilution and ab32572 anti-beta Catenin used at a 1/250 dilution. Opal Polymer HRP Ms + Rb was used as a secondary antibody.

  Panel A: merged staining of anti-ALDH1L1 (green; Opal^™520), anti-HIRA/HIR (magenta; Opal^™690) and anti-beta Catenin (grey; Opal^™570) on mouse liver.
  
  Panel B: anti-ALDH1L1 staining cytoplasm of hepatocytes in mouse liver.
  
  Panel C: anti-HIRA/HIR staining nucleus of hepatocytes in mouse liver.
  
  Panel D: anti-beta Catenin staining membrane of hepatocytes in mouse liver.
  
  Nuclear DNA was labeled with DAPI (shown in blue).

  The section was incubated in three rounds of staining: in the order of Anti-ALDH1L1 antibody [EPR25443-103] - Astrocyte Marker ab307696, Anti-HIRA/HIR antibody [EPR25299-11] ab302928 and ab32572 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• 

  Western blot - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

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

  The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 30806153).

  In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.

  In Western blot, Anti-beta Catenin antibody - Total protein control (ab32572) staining at 1/1000 dilution.

  All lanes: Western blot - Anti-beta Catenin (phospho S675) antibody [EPR28410-45] (Anti-beta Catenin (phospho S675) antibody [EPR28410-45] ab314450) at 1/1000 dilution

  Lane 1: Untreated SK-N-MC (human brain epithelial cell) whole cell lysate (untreated membrane) at 40 µg

  Lane 2: SK-N-MC treated with 100 uM forskolin for 30 minutes whole cell lysate (untreated membrane) at 40 µg

  Lane 3: Untreated SK-N-MC (human brain epithelial cell) whole cell lysate (Alkaline phosphatase treated membrane) at 40 µg

  Lane 4: SK-N-MC treated with 100 uM forskolin for 30 minutes whole cell lysate (Alkaline phosphatase treated membrane) at 40 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Observed band size: 85 kDa, 90 kDa, 36 kDa

  Exposure time: 180s

• 

  Western blot - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

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

  In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.

  In Western blot, Anti-beta Catenin antibody - Total protein control (ab32572) staining at 1/1000 dilution.

  All lanes: Western blot - Anti-beta Catenin (phospho S675) antibody [EPR28410-45] (Anti-beta Catenin (phospho S675) antibody [EPR28410-45] ab314450) at 1/1000 dilution

  Lane 1: Mouse colon tissue lysate (untreated membrane) at 20 µg

  Lane 2: Rat liver tissue lysate (untreated membrane) at 20 µg

  Lane 3: Mouse colon tissue lysate (Alkaline phosphatase treated membrane) at 20 µg

  Lane 4: Rat liver tissue lysate (Alkaline phosphatase treated membrane) at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 85 kDa, 90 kDa, 36 kDa

  Exposure time: 180s

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Immunohistochemical analysis of formalin fixed paraffin embedded human colon labelling beta Catenin with ab32572 at a concentration of 0.1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

  ab32572 anti-beta Catenin antibody [E247] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

  Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Immunohistochemical analysis of formalin fixed paraffin embedded human colon labelling beta Catenin with ab32572 at a concentration of 0.1µg/ml. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with a Bond™ Polymer Refine Detection kit. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution 2) for 20mins.

  ab32572 anti-beta Catenin antibody [E247] was incubated for 15mins at room temperature. Sections were counterstained with Hematoxylin. Image inset shows absence of staining in secondary antibody only control.

  Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• 

  Western blot - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.

  The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 30806153).

  In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.

  In Western blot, Anti-Catenin beta-1 antibody - Total protein control (ab32572) staining at 1/1000 dilution.

  All lanes: Western blot - Anti-beta Catenin (phospho S552) antibody [EPR28411-7] (Anti-beta Catenin (phospho S552) antibody [EPR28411-7] ab314502) at 1/1000 dilution

  Lane 1: NIH/3T3 (mouse embryonic fibroblast) starved for 24 hours whole cell lysate (untreated membrane) at 20 µg

  Lane 2: NIH/3T3 starved for 24 hours, then treated with 100 uM forskolin for 20 minutes whole cell lysate (untreated membrane) at 20 µg

  Lane 3: NIH/3T3 starved for 24 hours, then treated with 100 uM forskolin for 20 minutes whole cell lysate (Lambda phosphatase treated membrane) at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 85 kDa, 90 kDa, 36 kDa

  Exposure time: 103s

• 

  Western blot - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.

  The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 30806153).

  In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.

  In Western blot, Anti-Catenin beta-1 antibody - Total protein control (ab32572) staining at 1/1000 dilution.

  All lanes: Western blot - Anti-beta Catenin (phospho S552) antibody [EPR28411-7] (Anti-beta Catenin (phospho S552) antibody [EPR28411-7] ab314502) at 1/1000 dilution

  Lane 1: SW480 (human colorectal adenocarcinoma epithelial cell) whole cell lysate (untreated membrane) at 20 µg

  Lane 2: SW480 whole cell lysate (Lambda phosphatase membrane) at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Developed using the ECL technique.

  Observed band size: 85 kDa, 90 kDa, 36 kDa

  Exposure time: 136s

• 

  Western blot - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.

  The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 30806153).

  In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) staining at 1/200000 dilution.

  In Western blot, Anti-Catenin beta-1 antibody - Total protein control (ab32572) staining at 1/1000 dilution.

  All lanes: Western blot - Anti-beta Catenin (phospho S552) antibody [EPR28411-7] (Anti-beta Catenin (phospho S552) antibody [EPR28411-7] ab314502) at 1/1000 dilution

  Lane 1: Untreated SK-N-MC (human brain epithelial cell) whole cell lysate (untreated membrane) at 20 µg

  Lane 2: SK-N-MC treated with 100 uM forskolin for 30 minutes whole cell lysate (untreated membrane) at 20 µg

  Lane 3: SK-N-MC treated with 100 uM forskolin for 30 minutes whole cell lysate (Lambda phosphatase treated membrane) at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 85 kDa, 90 kDa, 36 kDa

  Exposure time: 15s

• 

  Immunocytochemistry - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  ab32572 staining of ß-catenin in a HCT116 cell spheroid. The cells were fixed with 4% formaldehyde (10 min), permeabilised with 0.5% Triton X-100 for 1h and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween overnight at room temperature. The spheroids were then incubated overnight at room temperature with ab32572 at 2 μg/ml and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, Mouse monoclonal [DM1A] to alpha Tubulin at 2 μg/ml. DAPI was used as nuclear counterstain (shown in blue). As secondary antibodies Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat anti-Rabbit (Alexa Fluor^® 488) (shown in green) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 Goat anti-Mouse (Alexa Fluor^® 594) (shown in magenta) were used, incubated overnight at room temperature. All permeabilization, blocking and antibody incubation steps were performed using a rotary shaker.

  Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

  The antibody ab32572 also worked using 100% methanol (5 min).

• 

  ChIP - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Chromatin was prepared from HCT 116 cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30 mins and then formaldehyde for 10 min.
  

  The ChIP was performed with 25 μg of chromatin, 5 μg of ab32572 (red), or 5 μg of rabbit normal IgG Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 (gray) and 20 μl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci)

  Primers and probes are from paper PMID: 28625518

  *http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

• 

  Western blot - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  False colour image of Western blot: Anti-beta Catenin antibody [E247] - ChIP Grade staining at 1/5000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32572 was shown to bind specifically to beta Catenin. A band was observed at 85/90 kDa in wild-type MCF7 cell lysates with no signal observed at this size in CTNNB1 knockout cell line Human CTNNB1 knockout MCF7 cell line ab286762. To generate this image, wild-type and CTNNB1 knockout MCF7 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

  All lanes: Western blot - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572) at 1/5000 dilution

  Lane 1: Wild-type MCF7 cell lysate at 20 µg

  Lane 2: CTNNB1 knockout MCF7 cell lysate, at 20 µg

  Lane 3: HeLa cell lysate at 20 µg

  Secondary

  All lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 85 kDa

  Observed band size: 85/90 kDa

• 

  Multiplex immunohistochemistry - Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572)

  Beta Catenin Multiplex immunohistochemistry staining of Rat liver using rabbit Anti-beta Catenin antibody

  Multiplex immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded rat liver tissue staining ALDH1L1 with Anti-ALDH1L1 antibody [EPR25443-103] - Astrocyte Marker ab307696 at a 1/5000 dilution, Anti-HIRA/HIR antibody [EPR25299-11] ab302928 anti-HIRA/HIR used at 1/100 dilution and ab32572 anti-beta Catenin used at a 1/250 dilution. Opal Polymer HRP Ms + Rb was used as a secondary antibody.

  Panel A: merged staining of anti-ALDH1L1 (green; Opal^™520), anti-HIRA/HIR (magenta; Opal^™690) and anti-beta Catenin (grey; Opal^™570) on rat liver.
  
  Panel B: anti-ALDH1L1 staining cytoplasm of hepatocytes in rat liver.
  
  Panel C: anti-HIRA/HIR staining nucleus of hepatocytes in rat liver.
  
  Panel D: anti-beta Catenin staining membrane of hepatocytes in rat liver.
  
  Nuclear DNA was labeled with DAPI (shown in blue).

  The section was incubated in three rounds of staining: in the order of Anti-ALDH1L1 antibody [EPR25443-103] - Astrocyte Marker ab307696, Anti-HIRA/HIR antibody [EPR25299-11] ab302928 and ab32572 for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.