All lanes : Anti-ATP5A antibody [EPR13030(B)] (ab176569) at 0.01 µg/ml

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : Mouse brain lysates
Lane 3 : Rat brain lysates

Lysates/proteins at 15 µg per lane.

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

Predicted band size: 60 kDa



Blocking and diluting buffer: 5% NFDM/TBST

Ab176569 (purified) staining ATP5A in HeLa (human cervix adenocarcinoma epithelial cell) by Immunocytochemistry/Immunofluorescence (ICC/IF). Cells were fixed with 4% paraformaldehyde and permeabilized n 0.1% TritonX-100. Samples were incubated with primary antibody at 1/500 dilution (4.2µg/ml).  An AlexaFluor^®488 Goat anti-Rabbit (ab150077) was used as a secondary antibody at 1/1000 dilution (2µg/ml). DAPI was used as a nuclear counterstain. Confocal image showing cytoplasmic staining in HeLa cells.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Rat kidney tissue sections labeling ATP5A with Purified ab176569 at 1:500 dilution (0.21 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody.Negative control:PBS instead of the primary antibody.Hematoxylin was used as a counterstain.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Mouse kidney tissue sections labeling ATP5A with Purified ab176569 at 1:500 dilution (0.21 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody.Negative control:PBS instead of the primary antibody.Hematoxylin was used as a counterstain.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human liver tissue sections labeling ATP5A with Purified ab176569 at 1:500 dilution (0.21 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use)was used as the secondary antibody.Negative control:PBS instead of the primary antibody.Hematoxylin was used as a counterstain.

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling ATP5A with purified ab176569 at 1:60 dilution (10 µg/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

All lanes : Anti-ATP5A antibody [EPR13030(B)] (ab176569) at 1/1000 dilution (unpurified)

Lane 1 : HepG2 cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : Human fetal liver lysate
Lane 4 : Human fetal lung lysate

Lysates/proteins at 10 µg per lane.

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

Developed using the ECL technique.

Predicted band size: 60 kDa

Immunofluorescence analysis of MCF7 cells labeling ATP5A using ab176569 (unpurified) at a 1/100 dilution (green). DAPI nuclear staining (blue).

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling ATP5A using ab176569 (unpurified) at a 1/50 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Human fetal heart tissue labeling ATP5A using ab176569 (unpurified) at a 1/50 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Flow cytometric analysis of permeabilized HeLa cells labeling ATP5A using ab176569 (unpurified) at a 1/10 dilution (red) or a rabbit IgG negative control (green).