Anti-CD73 antibody [EPR6114]

• IHC-P

Lab

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.

• IHC-P

Unknown

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.

• IHC-P

Unknown

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.

• IHC-P

Lab

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.

• WB

Lab

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] (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.

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 (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Lanes 1 - 4:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Observed band size: 70 kDa

false

• WB

Unknown

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

false

• WB

Lab

Western blot - Anti-CD73 antibody [EPR6114] (AB133582)

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.

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 2:

Western blot - Human NT5E knockout A-431 cell line (<a href='/en-us/products/cell-lines/human-nt5e-knockout-a-431-cell-line-ab261895'>ab261895</a>)

Lane 3:

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

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

false

• WB

Unknown

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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 63 kDa

false