All lanes : Anti-PODXL antibody [EPR9518] (ab150358) at 1/10000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : PODXL knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 58 kDa
Observed band size: 160 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab150358 observed at 160 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab150358 was shown to react with PODXL in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264984 (knockout cell lysate ab257210) was used. Wild-type HeLa and PODXL knockout HeLa 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. ab150358 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 10000 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.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue sections labeling PODXL with purified ab150358 at 1/1000 dilution (0.44 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) is the secondary antibody.

PBS instead of the primary antibody was used as the negative control.

All lanes : Anti-PODXL antibody [EPR9518] (ab150358) at 1/1000 dilution

Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : PODXL knockout HCT116 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 58 kDa
Observed band size: 200 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab150358 observed at 200 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

 ab150358 was shown to react with PODXL in wild-type HCT116 cells in western blot. Loss of signal was observed when knockout cell line ab266887 (knockout cell lysate ab257211) was used. Wild-Type HCT116 and PODXL knockout HCT116 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. ab150358 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) 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.

All lanes : Anti-PODXL antibody [EPR9518] (ab150358) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : PODXL knockout HAP1 whole cell lysate
Lane 3 : Raji whole cell lysate
Lane 4 : HeLa whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 58 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab150358 observed at 160 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab150358 was shown to specifically react with PODXL in wild-type cells as signal was lost in PODXL knockout cells. Wild-type and PODXL knockout samples were subjected to SDS-PAGE. Ab150358 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at a 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.

Anti-PODXL antibody [EPR9518] (ab150358) at 1/10000 dilution (purified) + Human fetal kidney lysates at 15 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/2000 dilution

Predicted band size: 58 kDa
Observed band size: 165 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer: 5% NFDM/TBST.

Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) cells labeling PODXL with purified ab150358 at 1/500. Cells were fixed with 100% methanol. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (ab150077) at 1/1000 dilution was used as the secondary antibody. Nuclei couterstained with DAPI (blue).

Secondary Only Control: PBS was used instead of the primary antibody as the negative control. 

All lanes : Anti-PODXL antibody [EPR9518] (ab150358) at 1/1000 dilution (unpurified)

Lane 1 : Raji lysate
Lane 2 : HeLa lysate
Lane 3 : Human fetal kidney lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 58 kDa
Observed band size: 165 kDa why is the actual band size different from the predicted?

Immunohistochemical analysis of paraffin embedded human kidney tissue labeling PODXL with unpurified ab150358 antibody at a dilution of 1/100. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling PODXL with purified ab150358 at 1/250 dilution (red). The secondary antibody was Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution. A Rabbit monoclonal IgG (Black) was used as the isotype control and cells without incubation with primary antibody and secondary antibody (Blue) were used as unlabeled control.

Unpurified ab150358 staining PODXL in human kidney tissue sections by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue samples were fixed with formaldehyde, cut into 20 micron slices, permeabilized with 0.05% tween-20 and blocked for 60 minutes at 25°C. Antigen retrieval was by heat mediation. The sample was incubated with primary antibody at a dilution of 1/250 at 25°C for 1 hour. An Alexa Fluor^® 488-conjugated donkey anti-rabbit polyclonal (1/1000) was used as the secondary antibody, at a dilution of 1/1200.

See Abreview

Immunohistochemical analysis of paraffin embedded human hepatocellular carcinoma vessels using unpurified ab150358 showing positive staining. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin embedded human breast adenocarcinoma tissue using unpurified ab150358 showing positive staining. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin embedded human endometrial carcinoma tissue using unpurified ab150358 showing positive staining. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin embedded human skeletal muscle tissue using unpurified ab150358 showing negative staining. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin embedded normal human kidney tissue using unpurified ab150358 showing positive staining. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin embedded human glioma tissue using unpurified ab150358 showing positive staining. Heat mediated antigen retrieval was performed before commencing with IHC staining protocol.