Product nameAnti-ATP5A antibody [15H4C4] - Mitochondrial Marker
See all ATP5A primary antibodies
DescriptionMouse monoclonal [15H4C4] to ATP5A - Mitochondrial Marker
Tested applicationsSuitable for: WB, ICC, IP, IHC-P, ICC/IF, Flow Cytmore details
Species reactivityReacts with: Mouse, Rat, Cow, Human, Pig, Caenorhabditis elegans, Drosophila melanogaster, Monkey
Tissue, cells or virus corresponding to Cow ATP5A. Purified mitochondrial Complex V (Cow).
- WB: Isolated mitochondria from human, cow, rat and mouse heart. Human liver tissue lysate. HepG2 whole cell lysate. ICC/IF: HeLa, MCF7 and MDA-MB-231 cells. IHC-P: Human heart tissue. Flow Cyt: HepG2 cells.
This antibody clone [15H4C4] is manufactured by Abcam.
We have the following conjugates available:
See other anti-mouse secondary antibodies that can be used with this antibody.
Storage instructionsShipped at 4°C. Store at +4°C.
Storage bufferPreservative: 0.02% Sodium azide
Constituent: HEPES buffered saline
Concentration information loading...
Purification notesNear homogeneity as judged by SDS-PAGE. The antibody was produced in vitro using hybridomas grown in serum-free medium, and then purified by biochemical fractionation.
Light chain typekappa
Our Abpromise guarantee covers the use of ab14748 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 53 kDa.|
|ICC||Use a concentration of 1 - 2 µg/ml.|
|IP||Use at 5 µg/mg of lysate.|
|IHC-P||Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.|
|ICC/IF||Use a concentration of 1 - 10 µg/ml.|
|Flow Cyt||Use a concentration of 1 µg/ml.
ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.
FunctionMitochondrial 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.
Tissue specificityFetal lung, heart, liver, gut and kidney. Expressed at higher levels in the fetal brain, retina and spinal cord.
Sequence similaritiesBelongs to the ATPase alpha/beta chains family.
modificationsThe N-terminus is blocked.
Cellular localizationMitochondrion inner membrane. Peripheral membrane protein.
- Information by UniProt
- ATP synthase alpha chain, mitochondrial antibody
- ATP synthase subunit alpha antibody
- ATP synthase subunit alpha mitochondrial antibody
Testes of bb8ms mutants show defects in post-meiotic, elongated spermatids.
(A-B) Spermatids from WT (A) and bb8ms (B) testis both have elongated cysts, but there are large spherical vesicles in the mutant (arrows) by phase contrast microscopy. Scale bars: 100 μm.
(C, D) Mitochondria of elongated spermatids stained with ATP5α antibody ab14748 in WT (C) and in bb8ms (D) mutants. ATP5α positive staining of the large vesicles in the cysts are indicated by arrow. Scale bars: 50 μm.
(E, F) JC-1 staining positive large vesicles (arrows) are absent from WT (E), but present in bb8ms elongated cysts (F). Scale bars: 25 μm.
Western blots were probed for HNE-damaged mitochondrial protein from brainstem (A), cerebellum (B) and “rest” brain (R). The sample designation indicates the age group (y for P25-P35, o for P45-P55), the genotype (KO, Ctrl for controls) and a number to distinguish independent samples.
Panel D is the blot from panel A reprobed for the mitochondrial marker ATPase (ATP5a) using ab14748 to demonstrate that extended sample storage did not degrade sample protein in general. Black lines in the MW lanes are magic marker on the film to indicate the positions of the prestained molecular weight standards on the blot.
For full image please see paper.
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 (Human liver hepatocellular carcinoma cell line) lysate at 20 µg
All lanes : Anti-ATP5A antibody [15H4C4] - Mitochondrial Marker (ab14748) at 1 µg/ml
Lane 1 : Human liver tissue lysate - total protein (ab29889)
Lane 2 : HepG2 (Human hepatocellular liver carcinoma cell line) whole cell lysate
Lysates/proteins at 10 µg per lane.
All lanes : 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.
ICC/IF image of ab14748 stained HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.
The cells were fixed in 100% methanol (5 minutes), 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 1 hour. The cells were then incubated overnight at +4°C with ab14748 at 1 µg/ml (shown in red) and ab6160 (Rat monoclonal to Tubulin) at 1 µg/ml (shown in green).
This was followed by an incubation at room temperature for 1 hour with ab150119, Goat Anti-Mouse IgG H&L (Alexa Fluor® 647) preadsorbed, at 0.5 µg/ml (shown in red) and ab150165, Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed, at 0.5 µg/ml (shown in green).
Nuclear DNA was labeled 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 minutes).
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 (Human breast adenocarcinoma cell line) cells.
The cells were fixed in 4% formaldehyde (10 minutes) and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab14748, 10 µg/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 1 hour. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1 hour. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43 µM.
ab14748 (1 µg/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 pH 6.1 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes.
Slides were counterstained with hematoxylin 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.
Overlay histogram showing HepG2 (Human liver hepatocellular carcinoma cell line) cells stained with ab14748 (red line).
The cells were fixed with 4% paraformaldehyde (10 minutes) and then permeabilized with 0.1% PBS-Tween for 20 minutes. 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, 1 µg/1x106 cells) for 30 minutes at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 minutes at 22ºC. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2 µg/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 minutes)/permeabilized in 0.1% PBS-Tween used under the same conditions.
This product has been referenced in:
- Yardeni T et al. High content image analysis reveals function of miR-124 upstream of Vimentin in regulating motor neuron mitochondria. Sci Rep 8:59 (2018). Read more (PubMed: 29311649) »
- Agnew T et al. MacroD1 Is a Promiscuous ADP-Ribosyl Hydrolase Localized to Mitochondria. Front Microbiol 9:20 (2018). Read more (PubMed: 29410655) »