Anti-CD73 antibody [EPR6114]

8 product images

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  Western blot - Anti-CD73 antibody [EPR6114] (ab133582)

  Lanes 1 - 3: Merged signal (red and green). Green - ab133582 observed at 63 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - 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 Anti-GAPDH antibody [6C5] - Loading Control 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 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 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

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

  Lane 1: Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

  Lane 2: NT5E knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

  Lane 3: A-375 (Human malignant melanoma cell line) whole cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 105 kDa, 143 kDa, 24 kDa, 35 kDa, 41 kDa, 44 kDa, 45 kDa, 53 kDa, 58 kDa, 61 kDa, 62 kDa, 63 kDa, 70 kDa, 88 kDa

  Observed band size: 104 kDa, 18 kDa, 55 kDa, 58 kDa

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  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 Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) 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.

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  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 Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) 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.

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  Western blot - Anti-CD73 antibody [EPR6114] (ab133582)

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

  All lanes: A375 ( Human malignant melanoma epithelial cell) whole cell lysates at 15 µg

  Secondary

  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: 63 kDa

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  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 Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) 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)

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

  Lane 1: Human fetal liver tissue lysate at 10 µg

  Lane 2: A375 cell lysate at 10 µg

  Lane 3: HepG2 cell lysate at 10 µg

  Secondary

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

  Predicted band size: 63 kDa

• 

  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)

  False colour image of Western blot: Anti-CD73 antibody [EPR6114] staining at 1/1000 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, 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 Human NT5E knockout A375 cell line ab281591 (knockout cell lysate Human NT5E knockout A375 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 (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-CD73 antibody [EPR6114] (ab133582) at 1/1000 dilution

  Lane 1: Wild-type A-375 cell lysate at 20 µg

  Lane 2: NT5E knockout A-375 cell lysate at 20 µg

  Lane 3: A431 cell lysate at 20 µg

  Lane 4: HepG2 cell lysate at 20 µg

  Secondary

  Lanes 1 - 4: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution

  Lanes 1 - 4: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution

  Performed under reducing conditions.

  Observed band size: 70 kDa