遺伝子ノックアウト細胞株 検証済
リコンビナント
RabMAb

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

製品の概要

  • 製品名

    Anti-beta Catenin antibody [E247] - ChIP Grade
    beta Catenin 一次抗体 製品一覧
  • 製品の詳細

    Rabbit monoclonal [E247] to beta Catenin - ChIP Grade
  • 由来種

    Rabbit
  • アプリケーション

    適用あり: IHC-Fr, IHC-P, WB, ICC/IF, IP, ChIPmore details
    適用なし: Flow Cyt
  • 種交差性

    交差種: Mouse, Rat, Sheep, Hamster, Cow, Human, Macaque monkey, African green monkey
  • 免疫原

    Synthetic peptide within Human beta Catenin aa 1-100 (N terminal). The exact sequence is proprietary.
    Database link: P35222

  • ポジティブ・コントロール

    • WB: A431, HeLa and wild-type HAP1 cell lysate. ICC/IF: A431 and wild-type HAP1 cells. SW480 and SK-N-SH cells. IHC-P: Human lung adenocarcinoma, kidney adenocarcinoma, colon adenocarcinoma, cervical carcinoma, breast carcinoma and papillary carcinoma of thyroid gland tissue; IP: A431 whole cell lysate.
  • 特記事項

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

製品の特性

アプリケーション

Our Abpromise guarantee covers the use of ab32572 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

アプリケーション Abreviews 特記事項
IHC-Fr 1/200.
IHC-P 1/500. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
WB 1/5000 - 1/10000. Detects a band of approximately 92 kDa (predicted molecular weight: 86 kDa).

We recommend Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773).

ICC/IF 1/250.

We recommend Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) secondary antibody.

IP 1/50.
ChIP Use at an assay dependent concentration.
  • 追加情報
    Is unsuitable for Flow Cyt.
  • ターゲット情報

    • 機能

      Key dowstream component of the canonical Wnt signaling pathway. 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. 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.
      Involved in the regulation of cell adhesion. The majority of beta-catenin is localized to the cell membrane and is part of E-cadherin/catenin adhesion complexes which are proposed to couple cadherins to the actin cytoskeleton.
    • 組織特異性

      Expressed in several hair follicle cell types: basal and peripheral matrix cells, and cells of the outer and inner root sheaths. Expressed in colon.
    • 関連疾患

      Defects in CTNNB1 are associated with colorectal cancer (CRC) [MIM:114500].
      Note=Activating mutations in CTNNB1 have oncogenic activity resulting in tumor development. Somatic mutations are found in various tumor types, including colon cancers, ovarian and prostate carcinomas, hepatoblastoma (HB), hepatocellular carcinoma (HCC). HBs are malignant embryonal tumors mainly affecting young children in the first three years of life.
      Defects in CTNNB1 are a cause of pilomatrixoma (PTR) [MIM:132600]; a common benign skin tumor.
      Defects in CTNNB1 are a cause of medulloblastoma (MDB) [MIM:155255]. MDB is a malignant, invasive embryonal tumor of the cerebellum with a preferential manifestation in children.
      Defects in CTNNB1 are a cause of susceptibility to ovarian cancer (OC) [MIM:167000]. Ovarian cancer common malignancy originating from ovarian tissue. Although many histologic types of ovarian neoplasms have been described, epithelial ovarian carcinoma is the most common form. Ovarian cancers are often asymptomatic and the recognized signs and symptoms, even of late-stage disease, are vague. Consequently, most patients are diagnosed with advanced disease.
      Note=A chromosomal aberration involving CTNNB1 is found in salivary gland pleiomorphic adenomas, the most common benign epithelial tumors of the salivary gland. Translocation t(3;8)(p21;q12) with PLAG1.
    • 配列類似性

      Belongs to the beta-catenin family.
      Contains 12 ARM repeats.
    • 翻訳後修飾

      Phosphorylation by GSK3B requires prior phosphorylation of Ser-45 by another kinase. Phosphorylation proceeds then from Thr-41 to Ser-37 and Ser-33.
      EGF stimulates tyrosine phosphorylation. Phosphorylation on Tyr-654 decreases CDH1 binding and enhances TBP binding.
      Ubiquitinated by the SCF(BTRC) E3 ligase complex when phosphorylated by GSK3B, leading to its degradation. Ubiquitinated by a E3 ubiquitin ligase complex containing UBE2D1, SIAH1, CACYBP/SIP, SKP1, APC and TBL1X, leading to its subsequent proteasomal degradation.
    • 細胞内局在

      Cytoplasm. Nucleus. Cytoplasm > cytoskeleton. Cell junction > adherens junction. Cell junction. Cell membrane. Cytoplasmic when it is unstabilized (high level of phosphorylation) or bound to CDH1. Translocates to the nucleus when it is stabilized (low level of phosphorylation). Interaction with GLIS2 and MUC1 promotes nuclear translocation. Interaction with EMD inhibits nuclear localization.
    • Information by UniProt
    • 参照データベース

    • 別名

      • b-catenin antibody
      • Beta catenin antibody
      • Beta-catenin antibody
      • Cadherin associated protein antibody
      • Catenin (cadherin associated protein), beta 1, 88kDa antibody
      • Catenin beta 1 antibody
      • Catenin beta-1 antibody
      • CATNB antibody
      • CHBCAT antibody
      • CTNB1_HUMAN antibody
      • CTNNB antibody
      • CTNNB1 antibody
      • DKFZp686D02253 antibody
      • FLJ25606 antibody
      • FLJ37923 antibody
      • OTTHUMP00000162082 antibody
      • OTTHUMP00000165222 antibody
      • OTTHUMP00000165223 antibody
      • OTTHUMP00000209288 antibody
      • OTTHUMP00000209289 antibody
      see all

    画像

    • Lane 1: Hap1 wildtype cell lysate (20 µg)

      Lane 2: CTNNB1 Hap1 knockout cell lysate (20 µg)

      Lane 3: HeLa wildtype cell lysate (20 µg)

      Lane 4: CTNNB1 HeLa knockout cell lysate (20 µg)

      Lanes 1 - 4: Merged signal (red and green). Green - ab32572 observed at 95 kDa. Red - loading control, ab8245 observed at 37 kDa.

      ab32572 was shown to react with beta Catenin in HeLa wildtype. Loss of signal was observed when knockout sample ab263756 was used. Wild-type and beta Catenin knockout samples were subjected to SDS-PAGE. ab32572 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 5000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and 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.
    • 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 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

    • Lane 1: Wild type HAP1 whole cell lysate (20 µg)
      Lane 2: CTNNB1 (β-catenin) knockout HAP1 whole cell lysate (20 µg)
      Lane 3: HeLa whole cell lysate (20 µg)
      Lane 4: A431 whole cell lysate (20 µg)

      Lanes 1 - 4: Merged signal (red and green). Green - ab32572 observed at 90 kDa. Red - 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 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 (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

    • 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 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 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).

    • Anti-beta Catenin antibody [E247] - ChIP Grade (ab32572) at 1/10000 dilution + A431 (Human epidermoid carcinoma epithelial cell) whole cell lysates at 15 µg

      Secondary
      Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

      Predicted band size: 86 kDa
      Observed band size: 92 kDa
      why is the actual band size different from the predicted?



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

      Exposure time: 180 seconds

    • 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) 

       

    • ab32572 staining beta Catenin in SW480 (Human colorectal adenocarcinoma cell line) cells treated with BIO (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 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 (ab96899) secondary antibody at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

    • 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) (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 (ab172730) instead of ab32572 in A431 whole cell lysate.

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

      Exposure time: 3 seconds

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



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

    • ab32572 staining beta Catenin in dog colon tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections).

      Tissue was fixed with formaldehyde and blocked with 10% serum for 30 minutes at 25°C; antigen retrieval was by heat mediation in 10 mM citrate buffer, pH6. Samples were incubated with primary antibody (1/250 in PBS with 1x casein) for 90 minutes at 25°C. A biotin-conjugated Goat anti-rabbit IgG polyclonal (1/200) was used as the secondary antibody.

      See Abreview

    • 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).

    • 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.

    • ab32572 at 1/200 staining mouse small intestine tissue sections by IHC-P.

      The tissue was formaldehyde fixed and a heat mediated antigen retrieval step was performed before incubation with the primary antibody. An HRP conjugated goat anti-rabbit antibody was used as the secondary.

      See Abreview

    • ab32572 staining beta Catenin in mouse liver tissue section by Immunohistochemistry (Frozen sections).

      Tissue samples were fixed with formaldehyde and blocked with 5% serum at 4°C for 30 minutes. The sample was incubated with primary antibody (1/200) in dilution buffer containing PBS and 3% goat serum at 4°C for 9 hours. An Alexa Fluor®488-conjugated Goat polyclonal to rabbit IgG was used as secondary antibody at 1/200 dilution.

      See Abreview

    • ab32572 staining human renal carcinoma tissue sections by IHC-P. 

      Sections were formaldehyde fixed and subjected to heat mediated antigen retrieval in citrate buffer (pH 6) prior to blocking with 1% milk for 45 minutes at 22°C.  The primary antibody was diluted 1/200 and incubated with the sample for 1 hour at 22°C.  An HRP conjugated goat anti-rabbit antibody, diluted 1/400, was used as the secondary.

      See Abreview

    • ab32572 staining beta Catenin in SK-N-SH (Human neuroblastoma cell line) cells treated with olanzapine (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 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 (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 the bEnd.5 murine cell line by ICC/IF (Immunocytochemistry/immunofluorescence).

      Cells were fixed with paraformaldehyde, permeabilized with 0.1% Triton in PBS and blocked with 10% serum for 30 minutes at 22°C. Samples were incubated with primary antibody (1/300) for 16 hours at 4°C. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody.

      See Abreview

    • 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.

    • 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.

      See Abreview

    参考文献

    This product has been referenced in:

    • Ma Z  et al. Autophagy promotes hepatic differentiation of hepatic progenitor cells by regulating the Wnt/ß-catenin signaling pathway. J Mol Histol 50:75-90 (2019). Read more (PubMed: 30604254) »
    • Wang W  et al. miR-410 acts as an oncogene in colorectal cancer cells by targeting dickkopf-related protein 1 via the Wnt/ß-catenin signaling pathway. Oncol Lett 17:807-814 (2019). Read more (PubMed: 30655833) »
    See all 345 Publications for this product

    レビューと Q&A

    1-3 of 3 Q&A

    Question
    Answer

    Unfortunately, we do not currently provide the blocking peptide for product Anti-beta Catenin antibody [E247] (ab32572).

    However, our full list of beta Catenin general protein and peptide products can be found here: beta Catenin Proteins and Peptides.

    Read More

    Answer

    Product Anti-beta Catenin antibody [E247] (ab32572) has unfortunately not yet been tested for use in ChIP applications, and is therefore not currently covered for use in ChIP by our Abpromise Guarantee.

    However, should you wish to try ab32572 for use in ChIP applications, this Ab may qualify for our Abtrial Testing Program.

    Additional ChIP resources can also be found here: ChIP Tips, Tricks and Troubleshooting.

    Read More

    Answer

    Antigen Retrieval may not always be necessary, but we do recommend Heat Induced Epitope Retrieval (HIER) for the majority of our Abs used in IHC applications. Our full protocol for HIER can be found here: IHC Antigen Retrieval Protocol.

    Read More

    Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
    For licensing inquiries, please contact partnerships@abcam.com

    登録