All lanes : Anti-P4HB antibody [EPR9499] (ab137110) at 1 µg/ml

Lane 1 : Wild-type HeLa whole cell lysate
Lane 2 : P4HB knockout HeLa whole cell lysate
Lane 3 : HEP G2 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 57 kDa

Lanes 1 - 3: Merged signal (red and green). Green - ab137110 observed at 57 kDa. Red - loading control, ab130007, observed at 125 kDa.

ab137110 was shown to specifically react with P4HB (Protein disulfide-isomerase) in wild-type HeLa cells as signal was lost in P4HB knockout cells. Wild-type and P4HB knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% milk. Ab137110 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1 μg/ml 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-P4HB antibody [EPR9499] (ab137110) at 1/10000 dilution (purified)

Lane 1 : HepG2 whole cell lysate
Lane 2 : HeLa whole cell lysate
Lane 3 : HEK293 whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 57 kDa
Observed band size: 57 kDa



Blocking and dilution buffer: 5% NFDM/TBST

All lanes : Anti-P4HB antibody [EPR9499] (ab137110) at 1/10000 dilution (purified)

Lane 1 : Raw264.7 whole cell lysate
Lane 2 : PC-12 whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 57 kDa
Observed band size: 57 kDa



Blocking and dilution buffer: 5% NFDM/TBST

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney carcinoma tissue labelling P4HB with purified ab137110 at a dilution of 1/1000. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse liver tissue labelling P4HB with purified ab137110 at a dilution of 1/1000. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat liver tissue labelling P4HB with purified ab137110 at a dilution of 1/1000. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling P4HB with purified ab137110 at a dilution of 1/150. Cells were fixed with 100% methanol and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1: primary antibody (1/150) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

Flow Cytometry analysis of HeLa cells labelling P4HB with purified ab137110 at a dilution of 1/300 (red). Cells were fixed with 4% paraformaldehyde. An Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

All lanes : Anti-P4HB antibody [EPR9499] (ab137110) at 1/1000 dilution (unpurified)

Lane 1 : HepG2 cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : 293T cell lysate
Lane 4 : A431 cell lysate

Lysates/proteins at 10 µg per lane.

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

Developed using the ECL technique.

Predicted band size: 57 kDa

All lanes : Anti-P4HB antibody [EPR9499] (ab137110) at 1/2000 dilution (unpurified)

Lane 1 : Mouse brain tissue lysate
Lane 2 : Mouse heart tissue lysate
Lane 3 : Mouse kidney tissue lysate
Lane 4 : Mouse spleen tissue lysate

Predicted band size: 57 kDa

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue labelling P4HB with unpurified ab137110 at a dilution of 1/100.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human brain tissue labelling P4HB with unpurified ab137110 at a dilution of 1/100.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunocytochemistry/Immunofluorescence analysis of HepG2 cells labelling P4HB (green) with unpurified ab137110 at a dilution of 1/250. Cell nuclei are shown in blue.

Overlay histogram showing HepG2 cells stained with unpurified ab137110 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (unpurified ab137110, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was a goat anti-rabbit Alexa Fluor^® 488 (IgG H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HepG2 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.