Anti-CD73 antibody [RM2050]

• Flow Cyt

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Flow Cytometry - Anti-CD73 antibody [RM2050] (AB317462)

Flow cytometric analysis of HeLa(human cervical adenocarcinoma epithelial cell, Left) / U-87 MG(human glioblastoma-astrocytoma epithelial cell, Right) cells labelling CD73 with ab317462 at 1/5000 dilution (0.01 ug)/Red compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Low expression : HeLa (PMID : 8566797)
Gated on viable cell.

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-CD73 antibody [RM2050] (AB317462)

Flow cytometric analysis of CD73 knockout A431(huam CD73 knock out human epidermoid carcinoma epithelial cell, Left) / Parental A431(Right) cells labelling CD73 with ab317462 at 1/500 dilution (0.1 ug)/Red compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Gated on viable cell.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD73 antibody [RM2050] (AB317462)

Immunohistochemical analysis of paraffin-embedded Human stomach tissue labeling CD73 with ab317462 at 1/100 (4.97 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Low expression : Weakly positive staining on blood vessels of human stomach. The section was incubated with ab317462 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD73 antibody [RM2050] (AB317462)

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling CD73 with ab317462 at 1/100 (4.97 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human spleen. The section was incubated with ab317462 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CD73 antibody [RM2050] (AB317462)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NT5E KO A431(human epidermoid carcinoma epithelial cell) cells labelling CD73 with ab317462 at 1/500 (0.994 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/mL dilution (Green).

Confocal image showing membrane and cytoplasmic staining in parental A431 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD73 antibody [RM2050] (AB317462)

Immunohistochemical analysis of paraffin-embedded Human liver cancer tissue labeling CD73 with ab317462 at 1/100 (4.97 ug/ml) dilution, followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Positive staining on human liver cancer (PMID : 30971294). The section was incubated with ab317462 for 30 mins at room temperature.
The immunostaining was performed on a Leica Biosystems BOND® RX instrument

Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IP

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Immunoprecipitation - Anti-CD73 antibody [RM2050] (AB317462)

CD73 was immunoprecipitated from 0.35 mg U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate with ab317462 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317462 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate
Lane 2 : ab317462 IP in U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab317462 in U-87 MG whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-CD73 antibody [RM2050] (ab317462) at 1/30 dilution

Lane 1:

U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate

Lane 2:

ab317462 IP in U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 32s

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-CD73 antibody [RM2050] (AB317462)

Flow cytometric analysis of Mouse spleen cells labelling CD73 with ab317462 at 1/50 dilution (1 ug)/Right compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Mouse spleen cell are co-stained with CD3 conjugated Alexa Fluor®647.
Gated on viable cell.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CD73 antibody [RM2050] (AB317462)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 4T1 (mouse mammary gland carcinoma epithelial cell) cells labelling CD73 with ab317462 at 1/500 (0.994 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/mL dilution (Green).

Confocal image showing membrane and cytoplasmic staining in 4T1 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labeled with DAPI (shown in blue).
Negative control : RAW 264.7 (PMID : 28060378)
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/mL dilution.

• Flow Cyt

Supplier Data

Flow Cytometry - Anti-CD73 antibody [RM2050] (AB317462)

Flow cytometric analysis of RAW 264.7(mouse Abelson murine leukemia virus-induced tumor macrophage, Left) / 4T1 (mouse mammary gland carcinoma epithelial cell, Right) cells labelling CD73 with ab317462 at 1/50 dilution (1 ug)/Red compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Negative control : RAW 264.7(PMID : 28060378)
Gated on viable cell.

• IP

Supplier Data

Immunoprecipitation - Anti-CD73 antibody [RM2050] (AB317462)

CD73 was immunoprecipitated from 0.35 mg 4T1 (mouse mammary gland carcinoma epithelial cell) whole cell lysate with ab317462 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab317462 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : 4T1 (mouse mammary gland carcinoma epithelial cell) whole cell lysate
Lane 2 : ab317462 IP in 4T1 (mouse mammary gland carcinoma epithelial cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab317462 in 4T1 whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-CD73 antibody [RM2050] (ab317462) at 1/30 dilution

Lane 1:

4T1 (mouse mammary gland carcinoma epithelial cell) whole cell lysate

Lane 2:

ab317462 IP in 4T1 (mouse mammary gland carcinoma epithelial cell) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-CD73 antibody [RM2050] (AB317462)

Negative control : HeLa and Raji (PMID : 8566797)

In Western blot, ab317462 was shown to bind specifically to CD73. A band was observed at 75 kDa in wild-type A431 cell lysates (lane 5) whereas loss of signal was observed in the CD73 knockout cell line (lane 6, ab261895/knockout cell lysate ab261704).

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-CD73 antibody [RM2050] (ab317462) at 1/1000 dilution

Lane 1:

U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate at 40 µg

Lane 2:

A375 (human malignant melanoma epithelial cell) whole cell lysate at 40 µg

Lane 3:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 40 µg

Lane 4:

Raji (human Burkitts lymphoma B lymphocyte) whole cell lysate at 40 µg

Lane 5:

Wild-type A431 (human epidermoid carcinoma epithelial cell) whole cell lysate at 40 µg

Lane 6:

CD73 knockout A431 whole cell lysate at 40 µg

Lane 7:

Human spleen tissue lysate at 40 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 1/100000 dilution

Observed band size: 75 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-CD73 antibody [RM2050] (AB317462)

Negative control : RAW264.7 (PMID : 28060378)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

All lanes:

Western blot - Anti-CD73 antibody [RM2050] (ab317462) at 1/1000 dilution

Lane 1:

4T1 (mouse mammary gland carcinoma epithelial cell) whole cell lysate at 40 µg

Lane 2:

RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 40 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

Observed band size: 75 kDa,36 kDa

false

Exposure time: 180s