The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Detects a band of approximately 53 kDa.
Use a concentration of 1 - 2 µg/ml.
Use at 5 µg/mg of lysate.
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Use a concentration of 1 - 10 µg/ml.
Use a concentration of 1 µg/ml.
ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.
Mitochondrial membrane ATP synthase (F(1)F(0) ATP synthase or Complex V) produces ATP from ADP in the presence of a proton gradient across the membrane which is generated by electron transport complexes of the respiratory chain. F-type ATPases consist of two structural domains, F(1) - containing the extramembraneous catalytic core, and F(0) - containing the membrane proton channel, linked together by a central stalk and a peripheral stalk. During catalysis, ATP synthesis in the catalytic domain of F(1) is coupled via a rotary mechanism of the central stalk subunits to proton translocation. Subunits alpha and beta form the catalytic core in F(1). Rotation of the central stalk against the surrounding alpha(3)beta(3) subunits leads to hydrolysis of ATP in three separate catalytic sites on the beta subunits. Subunit alpha does not bear the catalytic high-affinity ATP-binding sites.
Fetal lung, heart, liver, gut and kidney. Expressed at higher levels in the fetal brain, retina and spinal cord.
ICC/IF image of ab14748 stained HeLa cells. The cells were 100% methanol fixed (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab14748 at 1ug/ml (shown in red) and ab6160 (Rat monoclonal to Tubulin) at 1µg/ml (shown in green). This was followed by an incubation at room temperature for 1h with ab150119, Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed, at 0.5ug/ml (shown in red) and ab150165, Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed, at 0.5ug/ml (shown in green).
Nuclear DNA was labelled with DAPI (shown in blue).
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
This product also gave a positive signal under the same testing conditions in HeLa cells fixed with 4% formaldehyde (10 min).
Western blot - Anti-ATP5A antibody [15H4C4] - Mitochondrial Marker (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
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 - Anti-ATP5A antibody [15H4C4] - Mitochondrial Marker (ab14748)This image is courtesy of an anonymous Abreview
ab14748 staining ATP5A in MDA MB 231 cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with formaldehyde, permeabilized with 1% Triton X-100 and blocked with 10% BSA for 1 hour at 21°C. Samples were incubated with primary antibody (1/100 in BSA + 0.02% Tween 20) for 1 hour at 21°C. A DyLight® 550-conjugated goat anti-mouse IgG polyclonal (1/500) was used as the secondary antibody.
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.
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.