Overview

  • Product name
    Anti-E Cadherin (phospho S838 + S840) antibody [EP913(2)Y]
    See all E Cadherin primary antibodies
  • Description
    Rabbit monoclonal [EP913(2)Y] to E Cadherin (phospho S838 + S840)
  • Host species
    Rabbit
  • Specificity
    Detects E Cadherin phosphorylated at serine 838 and 840.
  • Tested applications
    Suitable for: WB, IP, IHC-Pmore details
    Unsuitable for: Flow Cyt or ICC
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human E Cadherin. The exact sequence is proprietary.

  • Positive control
    • WB: Human brain lysate; IHC-P: Human breast carcinoma or cervical carcinoma tissue.
  • General notes

     

     

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

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

Properties

Applications

Our Abpromise guarantee covers the use of ab76319 in the following tested applications.

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

Application Abreviews Notes
WB 1/500000. Predicted molecular weight: 97 kDa.
IP 1/50.
IHC-P 1/100. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

  • Application notes
    Is unsuitable for Flow Cyt or ICC.
  • Target

    • Function
      Cadherins are calcium-dependent cell adhesion proteins. They preferentially interact with themselves in a homophilic manner in connecting cells; cadherins may thus contribute to the sorting of heterogeneous cell types. CDH1 is involved in mechanisms regulating cell-cell adhesions, mobility and proliferation of epithelial cells. Has a potent invasive suppressor role. It is a ligand for integrin alpha-E/beta-7.
      E-Cad/CTF2 promotes non-amyloidogenic degradation of Abeta precursors. Has a strong inhibitory effect on APP C99 and C83 production.
    • Tissue specificity
      Non-neural epithelial tissues.
    • Involvement in disease
      Defects in CDH1 are the cause of hereditary diffuse gastric cancer (HDGC) [MIM:137215]. An autosomal dominant cancer predisposition syndrome with increased susceptibility to diffuse gastric cancer. Diffuse gastric cancer is a malignant disease characterized by poorly differentiated infiltrating lesions resulting in thickening of the stomach. Malignant tumors start in the stomach, can spread to the esophagus or the small intestine, and can extend through the stomach wall to nearby lymph nodes and organs. It also can metastasize to other parts of the body. Note=Heterozygous germline mutations CDH1 are responsible for familial cases of diffuse gastric cancer. Somatic mutations in the has also been found in patients with sporadic diffuse gastric cancer and lobular breast cancer.
      Defects in CDH1 are a cause of susceptibility to endometrial cancer (ENDMC) [MIM:608089].
      Defects in CDH1 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.
    • Sequence similarities
      Contains 5 cadherin domains.
    • Post-translational
      modifications
      During apoptosis or with calcium influx, cleaved by a membrane-bound metalloproteinase (ADAM10), PS1/gamma-secretase and caspase-3 to produce fragments of about 38 kDa (E-CAD/CTF1), 33 kDa (E-CAD/CTF2) and 29 kDa (E-CAD/CTF3), respectively. Processing by the metalloproteinase, induced by calcium influx, causes disruption of cell-cell adhesion and the subsequent release of beta-catenin into the cytoplasm. The residual membrane-tethered cleavage product is rapidly degraded via an intracellular proteolytic pathway. Cleavage by caspase-3 releases the cytoplasmic tail resulting in disintegration of the actin microfilament system. The gamma-secretase-mediated cleavage promotes disassembly of adherens junctions.
    • Cellular localization
      Cell junction. Cell membrane. Endosome. Golgi apparatus > trans-Golgi network. Colocalizes with DLGAP5 at sites of cell-cell contact in intestinal epithelial cells. Anchored to actin microfilaments through association with alpha-, beta- and gamma-catenin. Sequential proteolysis induced by apoptosis or calcium influx, results in translocation from sites of cell-cell contact to the cytoplasm. Colocalizes with RAB11A endosomes during its transport from the Golgi apparatus to the plasma membrane.
    • Information by UniProt
    • Database links
    • Alternative names
      • Arc 1 antibody
      • CADH1_HUMAN antibody
      • Cadherin 1 antibody
      • cadherin 1 type 1 E-cadherin antibody
      • Cadherin1 antibody
      • CAM 120/80 antibody
      • CD 324 antibody
      • CD324 antibody
      • CD324 antigen antibody
      • cdh1 antibody
      • CDHE antibody
      • E-Cad/CTF3 antibody
      • E-cadherin antibody
      • ECAD antibody
      • Epithelial cadherin antibody
      • epithelial calcium dependant adhesion protein antibody
      • LCAM antibody
      • Liver cell adhesion molecule antibody
      • UVO antibody
      • Uvomorulin antibody
      see all

    Images

    • Anti-E Cadherin (phospho S838 + S840) antibody [EP913(2)Y] (ab76319) at 1/500000 dilution (purified) + Mouse brain at 10 µg

      Secondary
      HRP goat anti-rabbit (H+L) at 1/1000 dilution

      Predicted band size: 97 kDa
      Observed band size: 145 kDa
      why is the actual band size different from the predicted?



      Blocking buffer: 5% NFDM/TBST

      Dilution buffer: 5% NFDM/TBST

    • Anti-E Cadherin (phospho S838 + S840) antibody [EP913(2)Y] (ab76319) at 1/500000 dilution (purified) + Rat brain at 10 µg

      Secondary
      HRP goat anti-rabbit (H+L) at 1/1000 dilution

      Predicted band size: 97 kDa
      Observed band size: 145 kDa why is the actual band size different from the predicted?



      Blocking buffer: 5% NFDM/TBST

      Dilution buffer: 5% NFDM/TBST

    • All lanes : Anti-E Cadherin (phospho S838 + S840) antibody [EP913(2)Y] (ab76319) at 1/500000 dilution (purified)

      Lane 1 : Untreated human fetal brain
      Lane 2 : Human fetal brain treated with alkaline phosphatase

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : HRP anti-rabbit IgG, specific to the non-reduced form of IgG at 1/1000 dilution

      Predicted band size: 97 kDa
      Observed band size: 145 kDa why is the actual band size different from the predicted?



      Blocking buffer: 5% NFDM/TBST

      Dilution buffer: 5% NFDM/TBST

    • Immunohistochemical staining of paraffin embedded human skin with purified ab76319 at a working dilution of 1 in 100. The secondary antibody used is a HRP polymer for rabbit IgG. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

    • ab76319 (purified) at 1/50 immunoprecipitating E cadherin in human fetal brain (Lane 1). For western blotting, a HRP-conjugated anti-rabbit IgG was used as the secondary antibody (1/1000).

      Blocking buffer and concentration: 5% NFDM/TBST.

      Diluting buffer and concentration: 5% NFDM /TBST.

    • All lanes : Anti-E Cadherin (phospho S838 + S840) antibody [EP913(2)Y] (ab76319) at 1/1000000 dilution (unpurified)

      Lane 1 : Human brain lysate, untreated
      Lane 2 : Human brain lysate treated with AP

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Goat anti-rabbit HRP at 1/1000 dilution

      Predicted band size: 97 kDa

    • Unpurified ab76319 staining E Cadherin in mouse skin (pilosebaceous untis) tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde, permeabilized with Tween-20 and blocked with 10% normal donkey serum + 1% serum for 40 minutes at room temperature; antigen retrieval was by heat mediation in a citrate buffer, pH 6. Samples were incubated with primary antibody (1/400 in 1% BSA) for 16 hours at 4°C. An Alexa Fluor® 488-conjugated donkey anti-rabbit IgG polyclonal (1/400) was used as the secondary antibody.

      See Abreview

    • Immunohistochemical analysis of paraffin-embedded human breast carcinoma using unpurified ab76319 at 1/100 dilution.

    • Immunohistochemical analysis of paraffin-embedded human cervical carcinoma using unpurified ab76319 at 1/100 dilution.

    References

    This product has been referenced in:
    • Xu XF  et al. Suppression of BMX-ARHGAP fusion gene inhibits epithelial-mesenchymal transition in gastric cancer cells via RhoA-mediated blockade of JAK/STAT axis. J Cell Biochem 120:439-451 (2019). Read more (PubMed: 30216523) »
    • Chiu CS  et al. Exploiting Honokiol-induced ER stress CHOP activation inhibits the growth and metastasis of melanoma by suppressing the MITF and ß-catenin pathways. Cancer Lett 442:113-125 (2019). Read more (PubMed: 30391358) »
    See all 18 Publications for this product

    Customer reviews and Q&As

    1-2 of 2 Abreviews or Q&A

    Application
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
    Blocking step
    BSA as blocking agent for 40 minute(s) · Concentration: 1% · Temperature: RT°C
    Antigen retrieval step
    Heat mediated - Buffer/Enzyme Used: citrate buffer pH6
    Sample
    Mouse Tissue sections (mouse skin, pilosebaceous units)
    Specification
    mouse skin, pilosebaceous units
    Permeabilization
    Yes - Tween-20
    Fixative
    Paraformaldehyde

    Abcam user community

    Verified customer

    Submitted Sep 15 2014

    Question
    Answer

    I've been following up on the issues that we spoke about earlier in regards to the Anti-E Cadherin (phospho S838 + S840) antibody. I have also received your corresponence and thank you for taking the time to send that to me. I'll try to answer all the questions that you have raised and try to get these antibodies working for you. "1. Could primary Ab be nonspecifically attached to plastic in 96 well plate, causing high background?" Although there is a possibility your findings that "Minimal difference between groups 1 and 2 – cells with or without primary antibodies, possibly because of high background intensity" leads me to believe that background problems are a result of the secondary antibody.   "2. How to reduce the intensity of background: extra wash after introducing  primary Ab, going beyond ABcam recommended dilution rate for the secondary Ab?" I would definitly recommend decreasing the concentration of the secondary antibody as well as blocking in 5% BSA for 1 hour at room temp.  "3. Should we try to use higher concentration of primary Ab to possibly engage more e-cadherin sites for secondary Ab to differentiate from the background? Could that also increase the background?" This may help I would recommend doing this if you still have target recognition issues but only after you have been able to reduce your background staining. "4. Do we really need to conduct the cell fixation, and could that cause the cell loss in the experiment?" We spoke of the need to perform fixation to preserve protein morpholoy and expose epitopes. It is very possible that changing the fixation to acetone or methanol will greatly help you experiments. "5. Could cell permealization add to the amount of reacting epitopes?" I've found that the epitope site recognized by this antibody is intracelluar. Permeablization, whether by a detergents such as NP-40 or Triton-X 100 is neccessary if the fixative does not permeablize (eg , formalin, pfa), the acetone fixation that we spoke of will permeablize the cells so a seperate step will no be needed. I've included a pdf detailing ICC fixation and permeablization steps for you (see attached). Here is the SwissProt page where I found that information: http://www.uniprot.org/uniprot/P12830 "6. Conducting the experiment on  glass to prevent possible non-specific secondary Ab  attachment  to plastic causing high background? Could that also contribute to the higher cell loss after fixation? Could that possibly improve the resolution?" Although glass may help , I would definitely work with changing other aspect of the protocol first. Here is a link to our standard ICC protocol as I have not found a product specific one: https://www.abcam.com/index.html?pageconfig=resource&rid=12129 I hope that these suggestions help. Please feel free to contact us with any other questions that you have.   

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