Anti-CD73 antibody [EPR28213-52]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-CD73 antibody [EPR28213-52] (AB313339)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NT5E KO A431 (human NT5E knockout epidermoid carcinoma epithelial cell, Left) / Parental A431(Right) cells labelling CD73 with ab313339 at 1/50 dilution (1 ug)/Red (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). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CD73 antibody [EPR28213-52] (AB313339)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NT5E KO A431 (NT5E knockout human epidermoid carcinoma epithelial cell) cells labelling CD73 with ab313339 at 1/50 (10.46 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green). Confocal image showing membranous staining in A431 cells and no staining in NT5E knockout A431 cells.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 (Red). 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 2ug/ml dilution.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CD73 antibody [EPR28213-52] (AB313339)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized U-87 MG (human glioblastoma-astrocytoma epithelial cell) cells labelling CD73 with ab313339 at 1/50 (10.46 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green). Confocal image showing membranous staining in U-87 MG cells.Negative control : HeLa (PMID : 8566797)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 (Red). 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 2ug/ml dilution.

• IP

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Immunoprecipitation - Anti-CD73 antibody [EPR28213-52] (AB313339)

CD73 was immunoprecipitated from 0.35 mg U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate with ab313339 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab313339 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 : ab313339 IP in U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab313339 in U-87 MG whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 180 seconds

All lanes:

Immunoprecipitation - Anti-CD73 antibody [EPR28213-52] (ab313339) at 1/30 dilution

All lanes:

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: 180s

• WB

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Western blot - Anti-CD73 antibody [EPR28213-52] (AB313339)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Low expression : HeLa and Raji(PMID : 8566797) In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 180 seconds

All lanes:

Western blot - Anti-CD73 antibody [EPR28213-52] (ab313339) at 1/1000 dilution

Lane 1:

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

Lane 2:

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

Lane 3:

Raji (human Burkitts lymphoma B lymphocyte) 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 1/100000 dilution

Observed band size: 68 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-CD73 antibody [EPR28213-52] (AB313339)

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

Performed under reducing conditions.

In Western blot, ab313339 was shown to bind specifically to CD73. A band was observed at 68 kDa in wild-type A431 cell lysates whereas loss of signal was observed in the CD73 knockout cell line.

In Western blot, anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/10000 dilution.

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

All lanes:

Western blot - Anti-CD73 antibody [EPR28213-52] (ab313339) at 1/1000 dilution

Lane 1:

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

Lane 2:

CD73 knockout A431 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 1/100000 dilution

Predicted band size: 63 kDa

Observed band size: 68 kDa

true

Exposure time: 92s

• WB

Lab

Western blot - Anti-CD73 antibody [EPR28213-52] (AB313339)

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

All lanes:

Western blot - Anti-CD73 antibody [EPR28213-52] (ab313339) at 1/1000 dilution

Lane 1:

A431 (Human epidermoid carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

A375 (Human malignant melanoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

Human spleen tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 63 kDa

Observed band size: 68 kDa

false

Exposure time: 60s