Overview

  • Product name
    Anti-ATP5A antibody [15H4C4] - Mitochondrial Marker
    See all ATP5A primary antibodies
  • Description
    Mouse monoclonal [15H4C4] to ATP5A - Mitochondrial Marker
  • Host species
    Mouse
  • Tested applications
    Suitable for: WB, ICC, IP, IHC-P, ICC/IF, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human, Pig, Caenorhabditis elegans, Drosophila melanogaster, Monkey
  • Immunogen

    Tissue, cells or virus corresponding to Cow ATP5A. Purified mitochondrial Complex V (Cow).

  • Positive control
    • 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.
  • General notes

    This antibody clone [15H4C4] is manufactured by Abcam.
    We have the following conjugates available:

    Anti-ATP5A antibody (FITC) [15H4C4] - Mitochondrial Marker (ab119688)

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

Properties

Applications

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.

Application Abreviews Notes
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.

Target

  • Function
    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.
  • Tissue specificity
    Fetal lung, heart, liver, gut and kidney. Expressed at higher levels in the fetal brain, retina and spinal cord.
  • Sequence similarities
    Belongs to the ATPase alpha/beta chains family.
  • Post-translational
    modifications
    The N-terminus is blocked.
  • Cellular localization
    Mitochondrion inner membrane. Peripheral membrane protein.
  • Information by UniProt
  • Database links
  • Alternative names
    • ATP synthase alpha chain, mitochondrial antibody
    • ATP synthase subunit alpha antibody
    • ATP synthase subunit alpha mitochondrial antibody
    • ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1, cardiac muscle antibody
    • ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit, 1 antibody
    • ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit, isoform 1, cardiac muscle antibody
    • ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit, isoform 2, non-cardiac muscle-like 2 antibody
    • ATP sythase (F1 ATPase) alpha subunit antibody
    • ATP5A antibody
    • Atp5a1 antibody
    • ATP5AL2 antibody
    • ATPA_HUMAN antibody
    • ATPM antibody
    • Epididymis secretory sperm binding protein Li 123m antibody
    • hATP1 antibody
    • HEL-S-123m antibody
    • MC5DN4 antibody
    • mitochondrial antibody
    • Mitochondrial ATP synthetase antibody
    • Mitochondrial ATP synthetase oligomycin resistant antibody
    • Modifier of Min 2 mouse homolog antibody
    • Modifier of Min 2, mouse, homolog of antibody
    • MOM2 antibody
    • OMR antibody
    • ORM antibody
    • OTTHUMP00000163475 antibody
    see all

Images

  • 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.

    Secondary
    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.

    See Abreview

  • 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.

References

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) »
See all 159 Publications for this product

Customer reviews and Q&As

1-10 of 30 Abreviews or Q&A

Application
Western blot
Sample
Mouse Tissue lysate - whole (Adipose Tissue)
Gel Running Conditions
Non-reduced Denaturing (1.5)
Loading amount
50 µg
Specification
Adipose Tissue
Blocking step
(agent) for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 30°C

Abcam user community

Verified customer

Submitted Aug 21 2018

Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (3T3)
Permeabilization
Yes - Acetone:Methanol
Specification
3T3
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 0.5% · Temperature: 4°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Aug 21 2018

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (brain tissue)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate ph 6
Permeabilization
No
Specification
brain tissue
Blocking step
Normal Goat Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 26°C
Fixative
Formaldehyde

Herr Dr. Markus Kipp

Verified customer

Submitted Aug 21 2018

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry
Sample
Mouse Cultured Cells (Cardiomyocytes)
Permeabilization
Yes - Triton x-100, 0.03%
Specification
Cardiomyocytes
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Mar 13 2018

Application
Immunocytochemistry
Sample
Rat Cultured Cells (Cardiomyocytes)
Permeabilization
Yes - Triton x-100, 0.03%
Specification
Cardiomyocytes
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Feb 22 2018

Application
Western blot
Sample
Human Cell lysate - whole cell (Vascular smooth muscle cells)
Gel Running Conditions
Reduced Denaturing
Loading amount
30 µg
Specification
Vascular smooth muscle cells
Blocking step
Milk as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 5µg/mL · Temperature: 21°C

Abcam user community

Verified customer

Submitted Nov 24 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - other (AML12 hepatocytes, mitochondrial fraction)
Gel Running Conditions
Reduced Denaturing
Loading amount
25 µg
Treatment
18
Specification
AML12 hepatocytes, mitochondrial fraction
Blocking step
BSA as blocking agent for 12 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Sep 29 2017

Abcam has not validated the combination of species/application used in this Abreview.
Application
Western blot
Sample
American Robin (Turdis migratorius) Cell lysate - other (fibroblasts, mitochondrial fraction)
Gel Running Conditions
Reduced Denaturing (18)
Loading amount
25 µg
Specification
fibroblasts, mitochondrial fraction
Blocking step
BSA as blocking agent for 12 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Sep 29 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (colon)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: citrite buffer, pH6.0
Specification
colon
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Aug 10 2016

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 21°C
Sample
Human Cell (MDA MB 231 cells)
Specification
MDA MB 231 cells
Permeabilization
Yes - 1% Triton
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Feb 19 2015

1-10 of 30 Abreviews or Q&A

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