Recombinant Anti-CD73 antibody [EPR28213-52] (ab313339)

All lanes : Anti-CD73 antibody [EPR28213-52] (ab313339) at 1/1000 dilution

Lane 1 : U-87 MG (human glioblastoma-astrocytoma epithelial cell) whole cell lysate
Lane 2 : Hela (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : Raji (human Burkitts lymphoma B lymphocyte) whole cell lysate

Lysates/proteins at 40 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 63 kDa
Observed band size: 68 kDa why is the actual band size different from the predicted?

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

Lane 1 : Wild-type A431 (human epidermoid carcinoma epithelial cell) whole cell lysate
Lane 2 : CD73 knockout A431 whole cell lysate

Lysates/proteins at 40 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 63 kDa
Observed band size: 68 kDa why is the actual band size different from the predicted?

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- Vinculin antibody (ab129002) loading control staining at 1/10000 dilution.

 

This blot was developed using a high sensitivity ECL substrate.

Exposure time: 92 seconds

 

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 µ g/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L ( Alexa Fluor ® 488) preadsorbed antibody at 1/1000 2 µ g/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 2.5 µ g/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 2 µ g/ml dilution.

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 µ g/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L ( Alexa Fluor ® 488) preadsorbed antibody at 1/1000 2 µ g/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 2.5 µ g/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 2 µ g/ml dilution.

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 µ g)/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.

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