Recombinant 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] (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.

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) 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) 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) 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.

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.