ATP Synthase Immunocapture Kit (ab109715)

1. We do not know the detail epitope of this antibody, it pulls down the full complex of the ATP synthase.
2. It is possible to target on the subunits of F1 fraction but very unlikely to IP the whole F1 fraction only with this product. Instead you may consider using Anti-ATP5A antibody [15H4C4] - Mitochondrial Marker (ab14748) https://www.abcam.com/atp5a-antibody-15h4c4-mitochondrial-marker-ab14748.html. This antibody specifically targets a subunit of the soluble fraction component ATP5A1 and has been tested in IP.
3. The solid volume of the beads with 500ug size is 50uL .

You are correct that you cannot use B-mercaptoethanol since it also reduces the disulfide bonds of IgG. The binding between antibodies and ATP synthase can be dissociate with SDS or Glycine buffer. These buffers will not affect disulfide bond but only elute the target complex from the beads and break the complex down to subunits. After eluting the ATP synthase subunits from the beads, the target proteins can be distributed on a gel without reducing agent. Only the co-eluted capture antibody (MW=150kDa), if there is any, remain on the top of the gel. We coat the beads with saturated ATP synthase antibody, therefore, it is possible that there might be some antibodies are not cross-linked to the beads.

The lab has confirmed that some IgG will come off (this is inevitable to some degree even though they are chemically cross linked to the beads), this can be minimized by washing the beads thoroughly after capture and by not using DTT in the SDS elution buffer (i.e. not regular SDSPAGE loading buffer + DTT). They also recommend to make sure that when running the gel, there is no DTT is in your SDS-PAGE buffer. This will ensure that any IgG that has been eluted remains at the top of the gel away from the bands of interest.

Thank you for your enquiry

Lauryl Maltoside is a non-ionic detergent which solubilize proteins but is able to keep the protein complex intact. MS501 immunocaptures ATP synthase as a complex, therefore, Lauryl Maltoside is a critical detergent to use with this kit (though sold separately). We do not suggest using another detergent since it may break the complex structure.



Please let us know if you have further questions.

Product code: 109714 Inquiry: Hi Abcam, Do you know if this kit will immunocapture the ATP synthase enzyme from C. elegans? Unlikely unfortunately – this kit was tested positive with human, rat and cow. Mouse was negative. I think it is unlikely to work with C.elegans. Product code: 109715: Inquiry: Hi Abcam, Do you know if this kit will capture ATP synthase from C. elegans? Thanks! This is the antibody for the above kit 109714. C.elegans is untested. Product code: 109907: Inquiry: Hi Abcam, The instructions to this kit says that you need to add the provided phospholipids or else the reaction will not work ("The phospholipids provided in this kit are essential for Complex V activity"). Why is this so? For example, are the phospholipids necessary for the correct folding of complex V? Thanks! Ab109714 and 109907 are similar products (109907 comes with sample and was designed for inhibition studies). Both kits require the addition of lipids. However you might anticipate that in the absence of lipids the enzyme would freely hydrolyze ATP, this does was not found to be the case.

Thank you for contacting us.

We do not currently offer the protein G- agarose beads used in this kit. Any commercially available protein-G agarose beads should work as control. An excellent resource for finding those is Biocompare.com.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.