Anti-ATP synthase Immunocapture antibody [12F4AD8AF8] (ab109867)

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.

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.

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.

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.

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.