Anti-EpCAM antibody [323/A3] (ab85987)

Mouse monoclonal EpCAM antibody [323/A3]. Validated in WB, IP, IHC, ICC, Flow Cyt and tested in Human. Cited in 7 publication(s). Immunogen corresponding to tissue, cells or virus.

Overview

  • Product name

    Anti-EpCAM antibody [323/A3]
    See all EpCAM primary antibodies
  • Description

    Mouse monoclonal [323/A3] to EpCAM
  • Host species

    Mouse
  • Specificity

    We have data to indicate that this antibody may not cross react with Rat. However, this has not been conclusively tested and expression levels may vary in certain cell lines/tissues.
  • Tested applications

    Suitable for: WB, IP, IHC-P, ICC, Flow Cytmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Tissue, cells or virus corresponding to Human EpCAM. MCF7 (human breast adenocarcinoma cell line) cells.

  • Positive control

    • WB: MCF7 cell lysate.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid repeated freeze / thaw cycles.
  • Storage buffer

    pH: 7.40
    Preservative: 0.097% Sodium azide
    Constituent: PBS
  • Concentration information loading...
  • Purity

    Protein A purified
  • Purification notes

    Purified from cell culture supernatant by protein A affinity chromatography, purity > 95% (by SDS-PAGE).
  • Clonality

    Monoclonal
  • Clone number

    323/A3
  • Isotype

    IgG1
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab85987 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 Use a concentration of 1 - 4 µg/ml. Predicted molecular weight: 35 kDa.
IP Use a concentration of 1 - 4 µg/ml. per 100-500ug of protein in 1ml lysate.
IHC-P Use a concentration of 1 - 10 µg/ml. Perform enzymatic antigen retrieval before commencing with IHC staining protocol.
ICC Use a concentration of 1 - 10 µg/ml.
Flow Cyt Use at an assay dependent concentration.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

Target

  • Function

    May act as a physical homophilic interaction molecule between intestinal epithelial cells (IECs) and intraepithelial lymphocytes (IELs) at the mucosal epithelium for providing immunological barrier as a first line of defense against mucosal infection. Plays a role in embryonic stem cells proliferation and differentiation. Up-regulates the expression of FABP5, MYC and cyclins A and E.
  • Tissue specificity

    Highly and selectively expressed by undifferentiated rather than differentiated embryonic stem cells (ESC). Levels rapidly diminish as soon as ESC's differentiate (at protein levels). Expressed in almost all epithelial cell membranes but not on mesodermal or neural cell membranes. Found on the surface of adenocarcinoma.
  • Involvement in disease

    Defects in EPCAM are the cause of diarrhea type 5 (DIAR5) [MIM:613217]. It is an intractable diarrhea of infancy characterized by villous atrophy and absence of inflammation, with intestinal epithelial cell dysplasia manifesting as focal epithelial tufts in the duodenum and jejunum.
    Defects in EPCAM are a cause of hereditary non-polyposis colorectal cancer type 8 (HNPCC8) [MIM:613244]. HNPCC is a disease associated with marked increase in cancer susceptibility. It is characterized by a familial predisposition to early-onset colorectal carcinoma (CRC) and extra-colonic tumors of the gastrointestinal, urological and female reproductive tracts. HNPCC is reported to be the most common form of inherited colorectal cancer in the Western world. Clinically, HNPCC is often divided into two subgroups. Type I is characterized by hereditary predisposition to colorectal cancer, a young age of onset, and carcinoma observed in the proximal colon. Type II is characterized by increased risk for cancers in certain tissues such as the uterus, ovary, breast, stomach, small intestine, skin, and larynx in addition to the colon. Diagnosis of classical HNPCC is based on the Amsterdam criteria: 3 or more relatives affected by colorectal cancer, one a first degree relative of the other two; 2 or more generation affected; 1 or more colorectal cancers presenting before 50 years of age; exclusion of hereditary polyposis syndromes. The term 'suspected HNPCC' or 'incomplete HNPCC' can be used to describe families who do not or only partially fulfill the Amsterdam criteria, but in whom a genetic basis for colon cancer is strongly suspected. Note=HNPCC8 results from heterozygous deletion of 3-prime exons of EPCAM and intergenic regions directly upstream of MSH2, resulting in transcriptional read-through and epigenetic silencing of MSH2 in tissues expressing EPCAM.
  • Sequence similarities

    Belongs to the EPCAM family.
    Contains 1 thyroglobulin type-1 domain.
  • Post-translational
    modifications

    Hyperglycosylated in carcinoma tissue as compared with autologous normal epithelia. Glycosylation at Asn-198 is crucial for protein stability.
  • Cellular localization

    Lateral cell membrane. Cell junction > tight junction. Co-localizes with CLDN7 at the lateral cell membrane and tight junction.
  • Information by UniProt
  • Database links

  • Alternative names

    • 17 1A antibody
    • 323/A3 antibody
    • Adenocarcinoma associated antigen antibody
    • Adenocarcinoma-associated antigen antibody
    • Antigen identified by monoclonal antibody AUA1 antibody
    • AUA1 antibody
    • CD326 antibody
    • CD326 antigen antibody
    • Cell surface glycoprotein Trop 1 antibody
    • Cell surface glycoprotein Trop 2 antibody
    • Cell surface glycoprotein Trop-1 antibody
    • CO 17A antibody
    • CO17 1A antibody
    • CO17A antibody
    • DIAR5 antibody
    • EGP 2 antibody
    • EGP antibody
    • EGP2 antibody
    • EGP314 antibody
    • EGP40 antibody
    • Ep CAM antibody
    • Ep-CAM antibody
    • EPCAM antibody
    • EPCAM_HUMAN antibody
    • EpCAM1 antibody
    • Epithelial cell adhesion molecule antibody
    • Epithelial Cell Adhesion Molecule Intracellular Domain (EpCAM-ICD) antibody
    • Epithelial cell surface antigen antibody
    • Epithelial cellular adhesion molecule antibody
    • Epithelial glycoprotein 1 antibody
    • Epithelial glycoprotein 314 antibody
    • Epithelial glycoprotein antibody
    • ESA antibody
    • GA733 1 antibody
    • GA733 2 antibody
    • GA733-2 antibody
    • gastrointestinal tumor-associated antigen 2, 35-KD glycoprotein antibody
    • gp4 antibody
    • hEGP 2 antibody
    • hEGP314 antibody
    • HNPCC8 antibody
    • Human epithelial glycoprotein 2 antibody
    • KS 1/4 antigen antibody
    • KS1/4 antibody
    • KSA antibody
    • Ly74 antibody
    • Lymphocyte antigen 74 antibody
    • M1S 1 antibody
    • M1S2 antibody
    • M4S1 antibody
    • Major gastrointestinal tumor associated protein GA733 2 antibody
    • Major gastrointestinal tumor-associated protein GA733-2 antibody
    • mEGP314 antibody
    • Membrane component chromosome 4 surface marker (35kD glycoprotein) antibody
    • Membrane component, chromosome 4, surface marker 1 antibody
    • Membrane component, chromosome 4, surface marker antibody
    • MIC18 antibody
    • MK 1 antibody
    • Protein 289A antibody
    • TACD1 antibody
    • TACSTD1 antibody
    • TROP1 antibody
    • Tumor associated calcium signal transducer 1 antibody
    • Tumor associated calcium signal transducer 2 precursor antibody
    • Tumor-associated calcium signal transducer 1 antibody
    see all

Images

  • Anti-EpCAM antibody [323/A3] (ab85987) at 2 µg/ml + MCF7 (human breast adenocarcinoma cell line) cell lysate

    Predicted band size: 35 kDa

References

This product has been referenced in:

  • Jin X  et al. Evaluation of Tumor-Derived Exosomal miRNA as Potential Diagnostic Biomarkers for Early-Stage Non-Small Cell Lung Cancer Using Next-Generation Sequencing. Clin Cancer Res 23:5311-5319 (2017). Read more (PubMed: 28606918) »
  • Winter MJ  et al. The epithelial cell adhesion molecule (Ep-CAM) as a morphoregulatory molecule is a tool in surgical pathology. Am J Pathol 163:2139-48 (2003). Read more (PubMed: 14633587) »
See all 8 Publications for this product

Customer reviews and Q&As

Question

Inquiry: 1. Order details: Batch number: lot: GR15581-2 Abcam product code: ab85987 Antibody storage conditions (temperature/reconstitution etc) -20 degree 2. Please describe the problem (high background, wrong band size, more bands, no band etc). Cannot detect the major band at 35 kDa (no band but some background) 3. On what material are you testing the antibody in WB? * Species: human * What’s cell line or tissue: ZR-75-1, breast cancer cell line * Cell extract or Nuclear extract: cell extract * Purified protein or Recombinant protein: whole cell lysate 3. The lysate How much protein was loaded: ˜50 ug What lysis buffer was used: Triton X-100 What protease inhibitors were used: sigma cocktail What loading buffer was used: SDS sample buffer (Laemmli buffer) Phosphatase inhibitors: NaF, Na3VO4, PMSF Did you heat the samples: temperature and time: 100 degree, 6 min 4. Electrophoresis/Gel conditions/ Transfer conditions Reducing or non reducing gel: reducing gel Reducing agent: DTT, beta-mercaptoethonal Gel percentage : 12% Transfer conditions: (Type of membrane, Protein transfer verified):PVDF, Tris-Glycine semi-dry transfer 5. Blocking conditions Buffer: TBS Blocking agent: milk, BSA, serum, what percentage: 5% non-fat milk in TBS with 0.1% Tween 20 Incubation time: 1h Incubation temperature: room temperature 6. Primary Antibody Species: mouse Reacts against: human * At what dilution(s) have you tested this antibody: 1:2,000 (3.5 uL/7mL) * What dilution buffer was used: 5% non-fat milk in TBS with 0.1% Tween 20 * Incubation time: over night (˜16 h) * Incubation temperature: 4 degree C * What washing steps were done: TBS with 0.1% Tween 20, 15 mins x3 7. Secondary Antibody Species: goat Reacts against: mouse At what dilution(s) have you tested this antibody: 1:10,000 Incubation time: 1.5 h Wash steps: TBS with 0.1% Tween 20, 15 mins x3 Fluorochrome or enzyme conjugate: HRP conjugated Do you know whether the problems you are experiencing come from the secondary? no, I have also tested the secondary antibody in a separate blot 8. Detection method ECl, ECl+, other detection method: ECL 9. Did you apply positive and negative controls along with the samples? Please specify. Human breast cancer cell line 10. Optimization attempts * How many times have you tried the Western? 2 times * Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): no * Do you obtain the same results every time e.g. are background bands always in the same place? no * What steps have you altered? I increase both primary and secondary antibody.

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Answer

Thank you again for sending your customer's protocol. The protocol looks correct, and my only concern is that the concentration of the antibody is lower than what is recommended on the datasheet, 1 - 4 ug/ml, or a dilution of 1/250 - 1/1000. Has the customer tried staining blots of these samples with any other antibodies?

I see bands on the customer's western blot that may represent Epcam, in particular the lower one, between the markers at 34 and 43 kDa. Epcam is glycosylated, and may run heavier than the predicted 35 kDa. For instance, a customer review of a different Epcam antibody, ab8601, shows a band that the customer believes represents Epcam in another breast cancer cell line, MCF7, at about 40 kDa. The review is at the following link:

https://www.abcam.com/index.html?datasheet=8601&tab=abreviews&intabreviewid=23069

However, I am not confident that what your customer is seeing is Epcam, and there are at least two bands in each lane, not one.

Is your customer interested in trying a different Epcam antibody, perhaps ab8601, or ab124825? I can send one of these, or a different one, as a free replacement.

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