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    products/primary-antibodies/cd73-antibody-epr6114-ab133582.pdf

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Cardiovascular Hypoxia Associated Proteins
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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-CD73 antibody [EPR6114] (ab133582)

  • Datasheet
  • SDS
Reviews (2) Submit a question References (10)

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Western blot - Anti-CD73 antibody [EPR6114] (ab133582)
  • Western blot - Anti-CD73 antibody [EPR6114] (ab133582)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD73 antibody [EPR6114] (ab133582)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD73 antibody [EPR6114] (ab133582)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD73 antibody [EPR6114] (ab133582)
  • Western blot - Anti-CD73 antibody [EPR6114] (ab133582)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD73 antibody [EPR6114] (ab133582)
  • Western blot - Anti-CD73 antibody [EPR6114] (ab133582)
  • Anti-CD73 antibody [EPR6114] (ab133582)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR6114] to CD73
  • Suitable for: IHC-P, WB
  • Knockout validated
  • Reacts with: Human

Conjugates logo Related conjugates and formulations

Carrier Free

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5’-Nucleotidase (CD73) Activity Assay Kit (Colorimetric) (ab235945)

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Overview

  • Product name

    Anti-CD73 antibody [EPR6114]
    See all CD73 primary antibodies
  • Description

    Rabbit monoclonal [EPR6114] to CD73
  • Host species

    Rabbit
  • Tested applications

    Suitable for: IHC-P, WBmore details
    Unsuitable for: Flow Cyt,ICC/IF or IP
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: Human fetal liver tissue lysate, Human liver, Human spleen; A375 and HepG2 whole cell lysate (ab7900) IHC-P: Human lung carcinoma, Human tonsil tissue
  • General notes

    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.

    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.

    Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at -20°C. Stable for 12 months at -20°C.
  • Storage buffer

    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: 40% Glycerol, PBS, 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EPR6114
  • Isotype

    IgG
  • Research areas

    • Cardiovascular
    • Hypoxia
    • Associated Proteins
    • Cardiovascular
    • Atherosclerosis
    • Vascular Inflammation
    • Leukocyte recruitment
    • Other

Associated products

  • Alternative Versions

    • Anti-CD73 antibody [EPR6114] - Low endotoxin, Azide free (ab227113)
    • Anti-CD73 antibody [EPR6114] - BSA and Azide free (ab271895)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
    • Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab97051)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • KO cell lines

    • Human NT5E knockout A-431 cell line (ab261895)
  • KO cell lysates

    • Human NT5E knockout A-431 cell lysate (ab261704)
  • Recombinant Protein

    • Recombinant mouse CD73 protein (His tag) (ab223023)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab133582 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
IHC-P
1/2000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

For unpurified use ad 1/100 - 1/250. See IHC antigen retrieval protocols.

WB (1)
1/1000 - 1/10000. Predicted molecular weight: 63 kDa.
Notes
IHC-P
1/2000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

For unpurified use ad 1/100 - 1/250. See IHC antigen retrieval protocols.

WB
1/1000 - 1/10000. Predicted molecular weight: 63 kDa.
Application notes
Is unsuitable for Flow Cyt,ICC/IF or IP.

Target

  • Function

    Hydrolyzes extracellular nucleotides into membrane permeable nucleosides.
  • Involvement in disease

    Defects in NT5E are the cause of calcification of joints and arteries (CAJA) [MIM:211800]. A condition characterized by adult-onset calcification of the lower extremity arteries, including the iliac, femoral and tibial arteries, and hand and foot capsule joints. Age of onset has been reported as early as the second decade of life, usually involving intense joint pain or calcification in the hands.
  • Sequence similarities

    Belongs to the 5'-nucleotidase family.
  • Cellular localization

    Cell membrane.
  • Target information above from: UniProt accession P21589 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 4907 Human
    • Omim: 129190 Human
    • SwissProt: P21589 Human
    • Unigene: 153952 Human
    • Alternative names

      • 5' NT antibody
      • 5' nucleotidase (CD73) antibody
      • 5' nucleotidase precursor antibody
      • 5' nucleotidase, ecto antibody
      • 5' nucleotidase, ecto (CD73) antibody
      • 5'-NT antibody
      • 5'-nucleotidase antibody
      • 5NTD_HUMAN antibody
      • CD73 antibody
      • CD73 antigen antibody
      • E5NT antibody
      • Ecto 5' nucleotidase antibody
      • Ecto-5'-nucleotidase antibody
      • eN antibody
      • eNT antibody
      • NT antibody
      • NT5 antibody
      • NT5E antibody
      • NTE antibody
      • Purine 5 Prime Nucleotidase antibody
      see all

    Images

    • Western blot - Anti-CD73 antibody [EPR6114] (ab133582)
      Western blot - Anti-CD73 antibody [EPR6114] (ab133582)
      All lanes : Anti-CD73 antibody [EPR6114] (ab133582) at 1/1000 dilution

      Lane 1 : Wild-type A-375 cell lysate
      Lane 2 : NT5E knockout A-375 cell lysate
      Lane 3 : A431 cell lysate
      Lane 4 : HepG2 cell lysate

      Lysates/proteins at 20 µg per lane.

      Performed under reducing conditions.

      Predicted band size: 63 kDa
      Observed band size: 70 kDa why is the actual band size different from the predicted?



      False colour image of Western blot: Anti-CD73 antibody [EPR6114] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab133582 was shown to bind specifically to CD73. A band was observed at 70 kDa in wild-type A-375 cell lysates with no signal observed at this size in NT5E knockout cell line ab281591 (knockout cell lysate ab282943). To generate this image, wild-type and NT5E knockout A-375 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution 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 (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

    • Western blot - Anti-CD73 antibody [EPR6114] (ab133582)
      Western blot - Anti-CD73 antibody [EPR6114] (ab133582)
      All lanes : Anti-CD73 antibody [EPR6114] (ab133582) at 1/1000 dilution

      Lane 1 : Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate
      Lane 2 : NT5E knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate
      Lane 3 : A-375 (Human malignant melanoma cell line) whole cell lysate

      Lysates/proteins at 20 µg per lane.

      Performed under reducing conditions.

      Predicted band size: 63 kDa



      Lanes 1 - 3: Merged signal (red and green). Green - ab133582 observed at 63 kDa. Red - loading control, ab8245, observed at 37 kDa.

      ab133582 was shown to recognize NT5E in wild-type A-431 cells as signal was lost at the expected MW in NT5E knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and NT5E knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab133582 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD73 antibody [EPR6114] (ab133582)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD73 antibody [EPR6114] (ab133582)

      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human spleen tissue sections labeling CD73 with Purified ab133582 at 1:2000 dilution (1.12 µg/ml).

      Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).

      ImmunoHistoProbe one step HRP Polymer (ready to use) was used.

      Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD73 antibody [EPR6114] (ab133582)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD73 antibody [EPR6114] (ab133582)

      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung carcinoma tissue sections labeling CD73 with Purified ab133582 at 1:2000 dilution (1.12 µg/ml).

      Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).

      ImmunoHistoProbe one step HRP Polymer (ready to use)was used.

      Negative control:PBS instead of the primary antibody.

      Hematoxylin was used as a counterstain.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD73 antibody [EPR6114] (ab133582)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD73 antibody [EPR6114] (ab133582)

      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human liver tissue sections labeling CD73 with Purified ab133582 at 1:2000 dilution (1.12 µg/ml).

      Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0).

      ImmunoHistoProbe one step HRP Polymer (ready to use) was used.

      Negative control: PBS instead of the primary antibody.

      Hematoxylin was used as a counterstain.

    • Western blot - Anti-CD73 antibody [EPR6114] (ab133582)
      Western blot - Anti-CD73 antibody [EPR6114] (ab133582)
      Anti-CD73 antibody [EPR6114] (ab133582) at 1/1000 dilution + A375 ( Human malignant melanoma epithelial cell) whole cell lysates at 15 µg

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

      Predicted band size: 63 kDa

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD73 antibody [EPR6114] (ab133582)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD73 antibody [EPR6114] (ab133582)

      Immunohistochemical analysis of paraffin-embedded human tonsil tissue labelling CD73 with ab133582 at 1/100 dilution.

      Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

    • Western blot - Anti-CD73 antibody [EPR6114] (ab133582)
      Western blot - Anti-CD73 antibody [EPR6114] (ab133582)
      All lanes : Anti-CD73 antibody [EPR6114] (ab133582) at 1/1000 dilution

      Lane 1 : Human fetal liver tissue lysate
      Lane 2 : A375 cell lysate
      Lane 3 : HepG2 cell lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : HRP labelled Goat anti-Rabbit IgG at 1/2000 dilution

      Predicted band size: 63 kDa

    • Anti-CD73 antibody [EPR6114] (ab133582)
      Anti-CD73 antibody [EPR6114] (ab133582)

    Protocols

    • Western blot protocols
    • Immunohistochemistry protocols
    • Immunocytochemistry & immunofluorescence protocols

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    References (10)

    Publishing research using ab133582? Please let us know so that we can cite the reference in this datasheet.

    ab133582 has been referenced in 10 publications.

    • Liu H  et al. Human amnion-derived mesenchymal stem cells improved the reproductive function of age-related diminished ovarian reserve in mice through Ampk/FoxO3a signaling pathway. Stem Cell Res Ther 12:317 (2021). PubMed: 34078462
    • Shrestha R  et al. TNF-a-mediated epithelial-to-mesenchymal transition regulates expression of immune checkpoint molecules in hepatocellular carcinoma. Mol Med Rep 21:1849-1860 (2020). PubMed: 32319631
    • Tsiampali J  et al. Enzymatic Activity of CD73 Modulates Invasion of Gliomas via Epithelial-Mesenchymal Transition-Like Reprogramming. Pharmaceuticals (Basel) 13:N/A (2020). PubMed: 33187081
    • Wangzhou K  et al. MiR-143-3p Inhibits Osteogenic Differentiation of Human Periodontal Ligament Cells by Targeting KLF5 and Inactivating the Wnt/ß-Catenin Pathway. Front Physiol 11:606967 (2020). PubMed: 33603676
    • Chen X  et al. Modulation of proliferation and differentiation of gingiva-derived mesenchymal stem cells by concentrated growth factors: Potential implications in tissue engineering for dental regeneration and repair. Int J Mol Med 44:37-46 (2019). PubMed: 31017269
    • Ikuta D  et al. Fibrosis in metastatic lymph nodes is clinically correlated to poor prognosis in colorectal cancer. Oncotarget 9:29574-29586 (2018). PubMed: 30038705
    • Kawasaki T  et al. Six1 is required for mouse dental follicle cell and human periodontal ligament-derived cell proliferation. Dev Growth Differ N/A:N/A (2016). IF . PubMed: 27241908
    • Zhang B  et al. The expression and clinical significance of CD73 molecule in human rectal adenocarcinoma. Tumour Biol 36:5459-66 (2015). IHC ; Human . PubMed: 25677906
    • Zhang HY  et al. Target tissue ectoenzyme CD39/CD73-expressing Foxp3+ regulatory T cells in patients with psoriasis. Clin Exp Dermatol 40:182-91 (2015). IHC ; Human . PubMed: 25284153
    • Chen SY  et al. Isolation and characterization of mesenchymal progenitor cells from human orbital adipose tissue. Invest Ophthalmol Vis Sci 55:4842-52 (2014). IF ; Human . PubMed: 24994870

    Customer reviews and Q&As

    Show All Reviews Q&A
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    1-2 of 2 Abreviews or Q&A

    Immunocytochemistry/ Immunofluorescence abreview for Anti-CD73 antibody [EPR6114]

    Poor
    Abreviews
    Abreviews
    This product is known to not work in this application or species.
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (Breast cancer cell line cells, spiked into human b)
    Permeabilization
    Yes - 0.1 % Triton X-100 in PBS
    Specification
    Breast cancer cell line cells, spiked into human b
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 20°C
    Fixative
    Paraformaldehyde
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Nov 16 2021

    Western blot abreview for Anti-CD73 antibody [EPR6114]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (T84)
    Gel Running Conditions
    Non-reduced Denaturing
    Loading amount
    1e+006 cells
    Specification
    T84
    Blocking step
    BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
    Read More
    The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.

    Abcam user community

    Verified customer

    Submitted Oct 19 2018

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