Overview

  • Product name
    Anti-ATP synthase Immunocapture antibody [12F4AD8AF8]
  • Description
    Mouse monoclonal [12F4AD8AF8] to ATP synthase Immunocapture
  • Host species
    Mouse
  • Tested applications
    Suitable for: WB, Flow Cyt, IPmore details
  • Species reactivity
    Reacts with: Mouse, Cow, Human
  • Immunogen

    Bovine ATP synthase

  • Positive control
  • General notes

    This antibody clone is manufactured by Abcam.

     

    ab109867 is a sample of pure immunocapture antibody, not immobilized to a solid support.

    Product was previously marketed under the MitoSciences sub-brand.

    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 ab109867 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 at an assay dependent concentration. PubMed: 22523357
Flow Cyt Use 1µg for 106 cells.

ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.

 

IP Use at an assay dependent concentration. 100 µg ab109867 can capture at least 50 µg ATP synthase Immunocapture (determined with 1 mg solubilized bovine heart mitochondria).

Target

  • Relevance
    Complex V, also called F1F0ATPase or ATP synthase, is responsible for ATP production in oxidative phosphorylation and can work in reverse as a proton pumping ATPase. The enzyme was thought to be localized exclusively to mitochondria. However, it has recently been identified on the plasma membrane of several cell types including hepatocytes where it functions as the HDL receptor, on endothelial cells where it may act as the angiostatin receptor, and on the surface of cancer cells. The enzyme in mammals is composed of 17 subunits, five of which make up the easily detached F1. The remainder subunits are components of two stalk domains and the proton pumping F0 part of the machinery. Two of the subunits of the F0 part are encoded on mitochondrial DNA while the other subunits are nuclear encoded. Mutations in the mitochondrial-encoded subunits of ATP synthase (Complex V) cause OXPHOS disease.

Images

  • Using the immunoprecipitation protocol provided, ATP synthase was isolated from various samples by antibody ab109867 crosslinked to protein G-agarose beads (ab109715). Protein bands identities were confirmed by mass spectrometry.
  • Overlay histogram showing HepG2 cells stained with ab109867 (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 (ab109867, 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 with 0.1% PBS-Tween for 20 min used under the same conditions.

References

This product has been referenced in:
  • Beutner G  et al. Cyclophilin D regulates the dynamic assembly of mitochondrial ATP synthase into synthasomes. Sci Rep 7:14488 (2017). Read more (PubMed: 29101324) »
  • Yan S  et al. F1F0 ATP Synthase-Cyclophilin D Interaction Contributes to Diabetes-Induced Synaptic Dysfunction and Cognitive Decline. Diabetes 65:3482-3494 (2016). Read more (PubMed: 27554467) »
See all 13 Publications for this product

Customer reviews and Q&As

1-7 of 7 Abreviews or Q&A

Application
Immunoprecipitation
Immuno-precipitation step
Protein A/G
Sample
Mouse Tissue lysate - whole (Brain mitochondria)
Specification
Brain mitochondria
Total protein in input
300 µg

Abcam user community

Verified customer

Submitted Jul 18 2014

Answer


The antibody was raised against the cow purified whole enzyme to maximize affinity for native active holoenzyme.

1) Our best immunocapture/immunoprecipitation antibodies bind the native folded proteins so often do not work in Western blot. This makes it very difficult to identify the subunit. Particularly if the binding site occurs at an interface between subunits such as ATP synthase alpha/beta subunits.

2) The antibody has been fully characterized for ATP synthase specificity and completeness of isolation in this paper: http://www.ncbi.nlm.nih.gov/pubmed/12110673

Specifically:

Immunocapture

Assays were run in 96-well plates (Falcon Probind) using 100 μl/well. The wells were prepared for immunocapture by first adsorbing goat anti-mouse IgG-Fc (IgG1+IgG2a+IgG2b+IgG3) at 5 μg/ml in TBS (50 mm Tris, pH 7.5, 150 mmNaCl) with 0.02% azide overnight at 4 °C and washed three times with TBS alone, incubated for 1 h at room temperature with the anti-ATPase capture mAb 12F4AD8AF8 (ab109867) at 5 μg/ml in TBS with 2.5% bovine serum albumin, and then washed four times with TBS. To control for nonspecific adsorption of ATPase, a nonspecific mouse monoclonal antibody was used as a null- capture mAb. Wells were then loaded with solubilized test samples diluted in TBS with 2.5% bovine serum albumin, incubated for 2 h at room temperature, and then washed four times with TBS.

Read More

Answer

Thank you for your reply.

I'm glad you found the information provided useful. You can use whole cell lysates with the antibody.However, it is critical to use LM as the detergent though. Other detergents such as NP40 or SDS in RIPA bufferwill break the complex. As an optional step, before addingLM, youcan sonicate the cell to disrupt the plasma membrane and break down the DNA, this step will ensure a better result.

Alternatively, you can use the mitochondrial fraction.

I hope this information has been of help. If you have any further questions, please do not hesitate to contact us again.

Read More

Answer

Thank you for contacting us and sorry for the delay in getting back to you.

I have contacted Mitosciences and they have shared the following protocols which can be used with the Anti-ATP synthase Immunocapture antibody [12F4AD8AF8] (ab109867).

Direct IP protocol 1:

prepare cell or tissue lysates using 1% LM as detergent;
incubate 1mg cell/tissue lysates with 2-5ug antibody for 30-2 hours at room temperature(RT) or longer at 4C;
Add 10ul agarose beads or proper volume of magnetic beads, incubate for another 30min-1 hour at RT.
Wash the beads three times with PBS/0.015%LM.
Elute the target protein with elution buffer (1%SDS or 0.2M Glycine pH2.0)



Direct IP protocol 2:

prepare cell or tissue lysates using 1% LM as detergent;
Dilute 1-5ug antibody in 1mll PBS or citrate phosphate buffer, incubate with 10ul agarose protein G beads or proper amount magnetic protein G beads for 30-2 hours at room temperature(RT);
Wash the beads three times with PBS.
Add 1mg cell/tissue lysates , incubate for another 30min-1 hour at RT.
Wash the beads three times with PBS/0.015%LM.
Elute the target protein with elution buffer (1%SDS or 0.2M Glycine pH2.0)



Cross-linking IP protocol:

Dilute 10-100ug antibody in 1ml PBS or citrate phosphate buffer, incubate with 10ul agarose protein G beads or proper amount magnetic protein G beads for 30-2 hours at room temperature(RT);
Wash the beads once with cross-linking buffer (0.2M sodium borate PH 8.0 or 0.2M triethanolamine pH8.0)
Incubate the beads with 1ml cross-linking reagent (20mM DMP in cross-linking buffer) for 30min at RT.
Wash the beads once with stop buffer (0.2M Ethanolamine or 1XTBST), then incubate in the same buffer for 30min at RT.
Antibody coated beads can be stored in PBS or TBS at 4C for up to 2 months.
prepare cell or tissue lysates using 1% LM as detergent;
incubate 1mg cell/tissue lysates with 10ul antibody coated beads for 30-2 hours at room temperature(RT) or longer at 4C;
Wash the beads three times with PBS/0.015%LM.
Elute the target protein with elution buffer (1%SDS or 0.2M Glycine pH2.0)



I hope this information is of help. We also ahve the directly conjugated ATP Synthase Immunocapture Kit (ab109715) which provides the antibody already directly conjugated to protein G-agarose beads. This can be purchased in three different sizes: 250, 500 and 750µg.

If youhave any further questions please do not hesitate to contact us again.

Read More
Abcam has not validated the combination of species/application used in this Abreview.
Application
IHC - Wholemount
Sample
Fruit fly (Drosophila melanogaster) Tissue (Dissected 3rd instar larvae)
Specification
Dissected 3rd instar larvae

Abcam user community

Verified customer

Submitted May 01 2012

Question
Answer

Thank you for your call today and for your interest in ab109867. Please see below for information about your testing discount code, and please let me know if you have any questions.

DISCOUNT CODE: ***
Expiration date: July 21, 2012

This code will give you 1 freeprimary antibodybefore the expiration date. To redeem this offer, please submit an Abreview for ab109867 in IHCand include this code in the “Additional Comments” section so we know the Abreview is for this promotion. For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.

The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code.

Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

Read More

Answer

Thank you for contacting us. The immunogen was not a recombinant protein.  It was purified ATP synthase enzyme from bovine heart (the whole complex).  Hence we don’t know the exact epitope to which the protein binds.  However, we do know for certain it bind to the F1 portion of the enzyme (confirmed by mass spectrometry analysis). I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.  

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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