Anti-ATP5A antibody [15H4C4] - Mitochondrial Marker (ab14748)

  Western blot - ATP5A antibody [15H4] (ab14748)

All lanes : Anti-ATP5A antibody [15H4C4] - Mitochondrial Marker (ab14748) at 1 µg/ml

Lane 1 : Isolated mitochondria from human heart at 10 µg
Lane 2 : Isolated mitochondria from bovine heart at 4 µg
Lane 3 : Isolated mitochondria from rat heart at 10 µg
Lane 4 : Isolated mitochondria from mouse heart at 10 µg
Lane 5 : HepG2 lysate at 20 µg

  Western blot - ATP5A antibody [15H4] (ab14748)

All lanes : Anti-ATP5A antibody [15H4C4] - Mitochondrial Marker (ab14748) at 1 µg/ml

Lane 1 : Liver (Human) Tissue Lysate - adult normal tissue (ab29889)
Lane 2 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Observed band size : 55 kDa (why is the actual band size different from the predicted?)
Additional bands at : 36 kDa. We are unsure as to the identity of these extra bands.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)-ATP5A antibody [15H4](ab14748)

ab14748 (1µg/ml) staining ATP5A in human heart (left ventricle), using an automated system (DAKO Autostainer Plus). Using this protocol there is mitochondrial staining of cardiomyocytes.
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH6.1 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.

  Immunocytochemistry/ Immunofluorescence - ATP5A antibody [15H4] (ab14748)

ICC/IF image of ab14748 stained MCF7 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab14748, 10µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  Flow Cytometry - ATP5A antibody [15H4] (ab14748)

Overlay histogram showing HepG2 cells stained with ab14748 (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 (ab14748, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HepG2 cells fixed with 80% methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.