All lanes : Anti-Poliovirus Receptor/PVR antibody [EPR17302] (ab205304) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : PVR knockout HEK-293T cell lysate
Lane 3 : Wild-type A549 cell lysate
Lane 4 : PVR knockout A549 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 45 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?

Lanes 1- 4: Merged signal (red and green). Green - ab205304 observed at 70 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab205304 was shown to react with Poliovirus Receptor/PVR in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266102 (knockout cell lysate ab257622) was used. Wild-type HEK-293T and PVR knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab205304 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Poliovirus Receptor/PVR was immunoprecipitated from 1mg of U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) whole cell lysate with ab205304 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab205304 at 1/2000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.
Lane 1: U-87 MG whole cell lysate 10µg (Input).
Lane 2: ab205304 IP in U-87 MG whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab205304 in U-87 MG whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 10 seconds.

All lanes : Anti-Poliovirus Receptor/PVR antibody [EPR17302] (ab205304) at 1/1000 dilution

Lane 1 : Wild-type A549 whole cell lysate
Lane 2 : PVR knockout A549 whole cell lysate
Lane 3 : U87-MG whole cell lysate
Lane 4 : HUVEC whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 45 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab205304 observed at 70 kDa (ab205304), 60-80 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab205304 was shown to specifically react with Poliovirus Receptor in wild-type A549 cells as signal was lost in PVR knockout cells. Wild-type and PVR knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab205304 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 : Anti-Poliovirus Receptor/PVR antibody [EPR17302] (ab205304) at 1/4000 dilution

Lane 1 : HT1080 (Human fibrosarcoma cell line) whole cell lysate
Lane 2 : U87-MG (Human glioblastoma-astrocytoma epithelial cell line) whole cell lysate
Lane 3 : HUVEC (Human umbilical vein endothelial cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Developed using the ECL technique.

Predicted band size: 45 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-Poliovirus Receptor/PVR antibody [EPR17302] (ab205304) at 1/1000 dilution + K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Developed using the ECL technique.

Predicted band size: 45 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Poliovirus Receptor/PVR antibody [EPR17302] (ab205304) at 1/1000 dilution

Lane 1 : Human fetal heart lysate
Lane 2 : Human fetal kidney lysate
Lane 3 : Human fetal spleen lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/10000 dilution

Developed using the ECL technique.

Predicted band size: 45 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Poliovirus Receptor/PVR antibody [EPR17302] (ab205304) at 1/1000 dilution

Lane 1 : Untreated K562 (Human chronic myelogenous leukemia cell line from bone marrow ) whole cell lysate
Lane 2 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate deglycosylation (PNGase F) treated

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

Developed using the ECL technique.

Predicted band size: 45 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.