Anti-beta Catenin antibody [E247] - ChIP Grade is a rabbit recombinant monoclonal antibody that is used to detect beta Catenin in ChIP, ICC/IF, IHC-P, IP, Western blot. Suitable for Human, Mouse, Rat samples.
- Specificity confirmed with CTNNB1 knockout cell line validation
- Using biophysical QC, antibody identity is confirmed at a molecular level for unrivaled batch-batch consistency
- Cited in over 1,070 publications
pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
mIHC | IHC-P | IP | ChIP | Flow Cyt | WB | ICC/IF | |
---|---|---|---|---|---|---|---|
Human | Expected | Tested | Tested | Tested | Not recommended | Tested | Tested |
Mouse | Tested | Tested | Tested | Expected | Not recommended | Expected | Not recommended |
Rat | Tested | Tested | Expected | Expected | Not recommended | Expected | Not recommended |
African green monkey | Predicted | Predicted | Predicted | Predicted | Not recommended | Predicted | Not recommended |
Cow | Predicted | Predicted | Predicted | Predicted | Not recommended | Predicted | Not recommended |
Hamster | Predicted | Predicted | Predicted | Predicted | Not recommended | Predicted | Not recommended |
Macaque monkey | Predicted | Predicted | Predicted | Predicted | Not recommended | Predicted | Not recommended |
Sheep | Predicted | Predicted | Predicted | Predicted | Not recommended | Predicted | Not recommended |
Species | Dilution info | Notes |
---|---|---|
Species Mouse | Dilution info 1/250 | Notes - |
Species Rat | Dilution info 1/250 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info Use at an assay dependent concentration. | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Sheep, Hamster, Cow, Macaque monkey, African green monkey | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse | Dilution info 1/500 | Notes Perform heat-mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. |
Species Rat | Dilution info 1/500 | Notes Perform heat-mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. |
Species Human | Dilution info 1/500 | Notes Perform heat-mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. |
Species | Dilution info | Notes |
---|---|---|
Species Sheep, Hamster, Cow, Macaque monkey, African green monkey | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse | Dilution info 1/30 | Notes - |
Species Human | Dilution info 1/30 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Rat | Dilution info Use at an assay dependent concentration. | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Sheep, Hamster, Cow, Macaque monkey, African green monkey | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse, Rat | Dilution info Use at an assay dependent concentration. | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Sheep, Hamster, Cow, Macaque monkey, African green monkey | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse, Rat, Human, Sheep, Hamster, Cow, Macaque monkey, African green monkey | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1/5000.00000 - 1/10000.00000 | Notes We recommend Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773). |
Species | Dilution info | Notes |
---|---|---|
Species Mouse, Rat | Dilution info Use at an assay dependent concentration. | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Sheep, Hamster, Cow, Macaque monkey, African green monkey | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1/250 | Notes We recommend Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) secondary antibody. |
Species | Dilution info | Notes |
---|---|---|
Species Rat | Dilution info - | Notes We recommend Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) secondary antibody. |
Species Mouse | Dilution info - | Notes We recommend Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) secondary antibody. |
Species Sheep | Dilution info - | Notes - |
Species Hamster | Dilution info - | Notes - |
Species Cow | Dilution info - | Notes - |
Species Macaque monkey | Dilution info - | Notes - |
Species African green monkey | Dilution info - | Notes - |
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Key downstream component of the canonical Wnt signaling pathway (PubMed:17524503, PubMed:18077326, PubMed:18086858, PubMed:18957423, PubMed:21262353, PubMed:22155184, PubMed:22647378, PubMed:22699938). In the absence of Wnt, forms a complex with AXIN1, AXIN2, APC, CSNK1A1 and GSK3B that promotes phosphorylation on N-terminal Ser and Thr residues and ubiquitination of CTNNB1 via BTRC and its subsequent degradation by the proteasome (PubMed:17524503, PubMed:18077326, PubMed:18086858, PubMed:18957423, PubMed:21262353, PubMed:22155184, PubMed:22647378, PubMed:22699938). In the presence of Wnt ligand, CTNNB1 is not ubiquitinated and accumulates in the nucleus, where it acts as a coactivator for transcription factors of the TCF/LEF family, leading to activate Wnt responsive genes (PubMed:17524503, PubMed:18077326, PubMed:18086858, PubMed:18957423, PubMed:21262353, PubMed:22155184, PubMed:22647378, PubMed:22699938). Involved in the regulation of cell adhesion, as component of an E-cadherin:catenin adhesion complex (By similarity). Acts as a negative regulator of centrosome cohesion (PubMed:18086858). Involved in the CDK2/PTPN6/CTNNB1/CEACAM1 pathway of insulin internalization (PubMed:21262353). Blocks anoikis of malignant kidney and intestinal epithelial cells and promotes their anchorage-independent growth by down-regulating DAPK2 (PubMed:18957423). Disrupts PML function and PML-NB formation by inhibiting RANBP2-mediated sumoylation of PML (PubMed:22155184). Promotes neurogenesis by maintaining sympathetic neuroblasts within the cell cycle (By similarity). Involved in chondrocyte differentiation via interaction with SOX9: SOX9-binding competes with the binding sites of TCF/LEF within CTNNB1, thereby inhibiting the Wnt signaling (By similarity). Acts as a positive regulator of odontoblast differentiation during mesenchymal tooth germ formation, via promoting the transcription of differentiation factors such as LEF1, BMP2 and BMP4 (By similarity). Activity is repressed in a MSX1-mediated manner at the bell stage of mesenchymal tooth germ formation which prevents premature differentiation of odontoblasts (By similarity).
CTNNB, OK/SW-cl.35, PRO2286, CTNNB1, Catenin beta-1, Beta-catenin
Anti-beta Catenin antibody [E247] - ChIP Grade is a rabbit recombinant monoclonal antibody that is used to detect beta Catenin in ChIP, ICC/IF, IHC-P, IP, Western blot. Suitable for Human, Mouse, Rat samples.
- Specificity confirmed with CTNNB1 knockout cell line validation
- Using biophysical QC, antibody identity is confirmed at a molecular level for unrivaled batch-batch consistency
- Cited in over 1,070 publications
pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
This antibody is not suitable for ICC testing in mouse and rat species.
Our inhouse testing indicated that this antibody does not work in Raw264.7 cell line in western blot. We have an alternative antibody ab68183 detecting weak band in lower expressor Raw264.7.
Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572) was developed by Abcam using patented rabbit monoclonal antibody technology and is validated for use in ChIP, ICC/IF, IHC-P, IP and WB.
Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572) was first used in a scientific publication in 1999 and has been cited over 1076 times in peer reviewed journals. It's performance in Western Blot in human, mouse and rat samples is trusted by the scientific community.
Abcam's high quality manufacturing and validation processes ensure Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572) has high sensitivity and specificity alongside high lot-to-lot consistency and reproducibility.
The specificity of Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572) has been confirmed by Western Blot testing in beta Catenin knockout HepG2 cells (Human CTNNB1 (beta Catenin) knockout Hep G2 cell line ab277911).
Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572) has 75 independent reviews from customers.
Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572) specifically detects beta Catenin (UniProt ID: P35222; Molecular weight: 86kDa) and is sold in a convenient trial size to enable initial testing (20 µL) and larger sizes for subsequent scaling up experiments (100 µL and 1 mL).
Conjugation-ready, carrier free format available for antibody clone E247 - Anti-beta Catenin antibody [E247] - BSA and Azide free ab196204.
Antibody clone E247 is also available pre-conjugated to a variety of labels for your convenience - Alexa Fluor® 488, Alexa Fluor® 647, HRP, Alexa Fluor® 594, Alexa Fluor® 555 (Alexa Fluor® 488 Anti-beta Catenin antibody [E247] ab194118, Alexa Fluor® 647 Anti-beta Catenin antibody [E247] ab194119, HRP Anti-beta Catenin antibody [E247] ab194120, Alexa Fluor® 594 Anti-beta Catenin antibody [E247] ab201771, Alexa Fluor® 555 Anti-beta Catenin antibody [E247] ab202496).
Top cited antibody on the market with >1370 citations. E247 is top rated with >75 reviews. Beta-catenin plays a crucial role in cell adhesion and gene transcription and its dysregulation is linked to various cancers and diseases including, myocardial infarction, arrhythmias, cardiomyopathie and Alzheimers disease
Patented technology
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
What are the advantages of a recombinant monoclonal antibody?
This product is a recombinant monoclonal antibody, which offers several advantages including:
For more information, read more on recombinant antibodies.
Beta Catenin also known by names such as CTNNB1 or beta-chip is an important protein involved in cell signaling and adhesion. This protein has a molecular weight of around 88 kDa. Beta Catenin is expressed in many cell types and tissues indicating its widespread role in various biological processes. It functions mechanically by mediating the linkage between cadherins and the actin cytoskeleton facilitating cell-cell adhesion. Beta Catenin is also a central part of transcription regulation processes in the nucleus.
This protein plays roles in both cell adhesion and the regulation of gene expression. Beta Catenin is a critical component of the Wnt signaling pathway where it can form complexes with other proteins to influence gene transcription. In the absence of Wnt signaling beta Catenin levels are low due to its degradation. However when the pathway is active it accumulates in the cytoplasm and eventually translocates to the nucleus where it interacts with TCF/LEF transcription factors to regulate the expression of target genes.
Beta Catenin plays a central role in the Wnt signaling pathway and influences cell fate decisions and cellular proliferation. It acts in concert with proteins such as Dishevelled (DVL) and Axin to coordinate these important biological processes. In the absence of Wnt signaling proteins such as APC and GSK-3β are responsible for beta Catenin degradation keeping its cellular levels in check. Beta Catenin's interaction with transcription factors in the nucleus makes it pivotal in the regulation of cell and tissue homeostasis.
Beta Catenin has associations with colorectal cancer and hepatocellular carcinoma. Its dysregulation can lead to unchecked cell proliferation and tumorigenesis. Often mutations in the beta Catenin gene (CTNNB1) or components of the Wnt pathway like APC are implicated in the development of these cancers. Its interplay with E-cadherin is important for maintaining tissue architecture and disruptions can lead to invasive cancer phenotypes. Understanding beta Catenin's role provides insights into therapeutic strategies for these cancers.
We have tested this species and application combination and it works. It is covered by our product promise.
We have not tested this specific species and application combination in-house, but expect it will work. It is covered by our product promise.
This species and application combination has not been tested, but we predict it will work based on strong homology. However, this combination is not covered by our product promise.
We do not recommend this combination. It is not covered by our product promise.
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Terms & Conditions.
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.
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
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
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
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
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
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
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).
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).
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
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
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
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).
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).
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
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).
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).
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
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
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.
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.
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
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
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)
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
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
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).
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).
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).
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.
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.
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
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
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
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
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)
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)
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
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
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
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
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
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
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).
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
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
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
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.
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