All lanes : Anti-Prohibitin antibody [EP2803Y] (ab75766) at 1/20000 dilution

Lane 1 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysates
Lane 2 : Mouse kidney tissue lysates
Lane 3 : Rat brain tissue lysates

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 30 kDa
Additional bands at: 30 kDa. We are unsure as to the identity of these extra bands.

ab75766 staining Prohibitin in rat kidney tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/500. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody.

Negative control 1: PBS in place of primary antibody.

ab75766 staining Prohibitin in NIH/3T3 (mouse embryo fibroblast cell line) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody. ab7291 (1/1000) and ab150120 (1/1000) were used as counterstains for primary antibody ab75766 and secondary antibody ab150077 respectively and DAPI was used as a nuclear counterstain.

Negative control 1: Rabbit primary antibody and anti-mouse secondary antibody (ab150120)
Negative control 2: Mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab150077)

Overlay histogram showing HepG2 (human liver hepatocellular carcinoma cell line) cells stained with ab75766 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab75766, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit 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 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

ab75766 immunoprecipitating Prohibitin. 10µg of cell lysate was incubated with primary antibody at a dilution of 1/40 and VeriBlot for IP Detection Reagent (HRP) (ab131366) at a dilution of 1/10000.

Lane 1: HEK-293 (human epithelial cell line from embryonic kidney) whole cell lysate (10ug)
Lane 2: HEK-293 whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of  ab75766 in HEK-293 whole cell lysate

All lanes : Anti-Prohibitin antibody [EP2803Y] (ab75766) at 1/20000 dilution

Lane 1 : MCF7 (human breast adenocarcinoma cell line) whole cell lysates
Lane 2 : A431 (human epidermoid carcinoma cell line) whole cell lysates
Lane 3 : HEK-293 (human epithelial cell line from embryonic kidney) whole cell lysates

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 30 kDa
Additional bands at: 30 kDa. We are unsure as to the identity of these extra bands.

All lanes : Anti-Prohibitin antibody [EP2803Y] (ab75766) at 1/20000 dilution

Lane 1 : MCF7 (human breast adenocarcinoma cell line) cell lysate
Lane 2 : A431 (human epidermoid carcinoma cell line) cell lysate
Lane 3 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cell lysate
Lane 4 : Ramos (human Burkitt's lymphoma cell line) cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 30 kDa
Observed band size: 30 kDa

ab75766 staining Prohibitin in mouse kidney tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/500. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody.

Negative control 1: PBS in place of primary antibody.

ab75766 staining Prohibitin in human liver tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/500. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody.

Negative control 1: PBS in place of primary antibody.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse kidney tissue labelling Prohibitin with ab75766 at 1/250.

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

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse stomach tissue labelling Prohibitin with ab75766 at 1/250.

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

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse liver tissue labelling Prohibitin with ab75766 at 1/250.

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

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat liver tissue labelling Prohibitin with ab75766 at 1/250.

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

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat stomach tissue labelling Prohibitin with ab75766 at 1/250.

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

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of normal liver tissue labelling Prohibitin with ab75766.

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

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of normal placenta tissue labelling Prohibitin with ab75766.

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

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of breast carcinoma tissue labelling Prohibitin with ab75766.

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

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue labelling Prohibitin with ab75766 at 1/250.

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

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human hepatocellular tissue labelling Prohibitin with ab75766 at 1/250.

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

Immunocytochemistry/Immunofluorescence analysis of HeLa (human epithelial cell line from cervix adenocarcinoma) cells labelling Prohibitin with ab75766 at 1/100. An Alexa Fluor^® 488-conjugated anti-rabbit IgG was used as the secondary antibody.

Immunocytochemistry/Immunofluorescence analysis of NIH/3T3 (mouse embryo fibroblast cell line) cells labelling Prohibitin with ab75766 at 1/100. An Alexa Fluor^® 488-conjugated anti-rabbit IgG was used as the secondary antibody.

ab75766 staining Prohibitin in the human cell line A431 (human epidermoid carcinoma cell line) by flow cytometry. Cells were fixed with 4% paraformaldehyde and the sample was incubated with the primary antibody at a dilution of 1/120. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/500 was used as the secondary antibody.

Isoytype control: Rabbit monoclonal IgG (Black)

Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)