Overview

  • Product name

    MitoTox™ Complex V OXPHOS Activity Assay Kit
    See all Complex V kits
  • Detection method

    Colorimetric
  • Sample type

    Inhibitor compounds
  • Assay type

    Direct
  • Assay time

    4h 00m
  • Species reactivity

    Reacts with: Rat, Cow, Human
    Does not react with: Mouse
  • Product overview

    MitoTox™ Complex V OXPHOS Activity Assay Kit (ab109907) is designed for testing the direct inhibitory effect of compounds on Complex V activity in only 4 hours. Complex V extracted from the provided bovine heart mitochondria (a rich source of Complex V) is immunocaptured by specific antibodies on the plate. Complex V activity can be observed as decrease in absorbance at OD 340 nm. The intra-assay and inter-assay variation of this assay are both < 10%.


    Inhibitory effects of compounds on Complex I activity can be tested in two different ways: 1. Screening format, where up to 23 compounds can be tested at a single concentration in triplicate; 2. Dose response (IC50) format, where two compounds known to affect Complex V activity can be tested at 11 different data points in triplicate.


    Testing for mitochondrial function has become a key aspect of drug discovery. Mitochondria can be affected by drug treatment, resulting into cardio- and hepatotoxic side effects that can lead to drug withdrawal from the market. Therefore, there is increasing emphasis on testing the impact on mitochondria early on in the drug development process to reduce failure rates during preclinical and clinical phases. 

  • Notes

    Store Phospholipids, Bovine heart mitochondria and Activity Buffer at -80°C. Store all other components at 4°C.

    Related products

    Review the mitochondrial assay guide, or the full metabolism assay guide to learn about more assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress, and also how to assay metabolic function in live cells using your plate reader.

  • Platform

    Microplate reader

Properties

Images

  • Dose response curve for aurovertin, a non-competitive inhibitor of Complex V.

  • Typical dose response curve for oligomycin. Assay was performed following the Dose Response Assay Procedure using oligomycin, a well known Complex V inhibitor. Oligomycin was prepared in DMSO to generate a 10 mM stock. Starting with a 50 µM final concentration in well, 1:10 serial dilutions of oligomycin were generated.

Protocols

References

This product has been referenced in:

  • Goldberg J  et al. The mitochondrial ATP synthase is a shared drug target for aging and dementia. Aging Cell 17:N/A (2018). Mouse . Read more (PubMed: 29316249) »
  • Kim YR  et al. Toxoplasma gondii GRA8 induces ATP5A1-SIRT3-mediated mitochondrial metabolic resuscitation: a potential therapy for sepsis. Exp Mol Med 50:e464 (2018). Read more (PubMed: 29869623) »
See all 9 Publications for this product

Customer reviews and Q&As

1-7 of 7 Abreviews or Q&A

Question
Answer

Unfortunately not. It comes with a sample (bovine) which cannot be substituted for mouse.

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Answer

Thank you for contacting us.

Unfortunately none of our products measure the forward reaction. As far as I know there are no companies selling kits measuring the forward reaction of the ATP synthase I am afraid.

I am sorry I could not be more helpful. Please do not hesitate to contract us if you need further advice or information.

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Answer

The kits that are the best suited for your experiment are listed below. I am having our datasheets team update the species reactivity.
ab109903, Complex I; reactivity = Human, mouse, rat, bovine
https://www.abcam.com/mitotox%E2%84%A2-complex-i-oxphos-activity-microplate-assay-ab109903.html
ab109904, Complex II, reactivity = Human, mouse and bovine
https://www.abcam.com/mitotox%E2%84%A2-complex-ii-oxphos-activity-microplate-assay-ab109904.html
ab109905, Complex II + III, reactivity = This is an in solution assay so it will work with mitochondria from any species, provided that the mitochondria is of good quality. It will not work with homogenate – it requires purified mitochondria
https://www.abcam.com/mitotoxtrade-complex-ii-nbsp-iii-oxphos-activity-microplate-assay-ab109905.html
ab109906, Complex IV, reactivity = Human and bovine
https://www.abcam.com/mitotoxtrade-complex-iv-oxphos-activity-microplate-assay-ab109906.html
ab109907, Complex V, reactivity = Human, mouse, rat and bovine.
https://www.abcam.com/mitotoxtrade-complex-v-oxphos-activity-microplate-assay-ab109907.html

ab110419, Complexes I - V (contains the five kits above at a discounted price)
https://www.abcam.com/mitotox%E2%84%A2-complete-oxphos-activity-assay-kit-5-assays-ab110419.html
In regards to inhibitor treatment, the laboratory agrees that the effects would be reversible. See their response and recommendations below:

If you were to treat the cells with for example rotenone, then isolate the mitochondria from rotenone treated cells, this will wash off all the rotenone from the sample during the sample prep and would simply find no inhibition detected by the assay.

What we would recommend is to test normal mitochondria in-vitro with the inhibitor compound as it is suggested in the kit protocols above.
The kits have a bovine heart mitochondria control, which you can use to see how the assay works (creating a positive and negative control with BHM plus or minus inhibitors).

Then you can compare the BHM results with the results obtained with the mitochondria prepared from your cell system.
The compound inhibitor must be present during the activity assay, otherwise the data will not reflect the immediate in vitro inhibitory effect of the compound, but more likely the downstream effect of that compound (i.e. If the compound generates as a downstream effect oxidative stress and this in turn affects the activity of one of the complexes of the electron transport chain).

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Answer

Thank you for contacting us.

The bovine mitochondria is in Tris, sucrose, EDTA buffer pH 7.8


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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Answer

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.

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Answer

The rate quoted in the booklet is mOD/min. I think this customer is quoting OD. Therefore 0.12/18 is 0.0067 OD/min = 6.7 mOD/min

Changing the window analyzed to identify the steepest straight line slope is necessary and then to compare all samples over this time period.








I hope this is helpful. Please contact us again if you have any further questions.

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Answer

Thank you for your enquiry.

The negative control chosen for ab109907 MitoTox™ Complex V OXPHOS Activity Microplate Assaywould depend on whatyou want to accomplish and obtain from the experiment.

Some people test the specificity of the assay by testing specific and non-specific inhibitors of the electron transport chain. For example, you could test for MTOX5 oligomycin as positive control andantimycin as negative control (which is specific inhibitor for complex III and will not inhibit complex V). The dose for the negative control is important,our suggestion is 10 uM.

However if the interest is not specificity but simply a compound that has no effect in any of the complexes, but it is known to affect mitochondria by a different mechanism then we would suggest using chloramphenicol (10 – 50 uM).Thisshould not affect any of the MTOX assay kits.

There are many others we could suggest, but without knowing exactly what the aim of your negative control is, it is difficult to do so.

Other options could be dinitrophenol or FCCP. In an invitro system like the MTOX assays DNP and FCCP will not affect the activity of the enzymes. However, in a cultured system DNP and FCCP will uncouple mitochondria.

I hope this will be helpful. if you require any further information in this case, please do not hesitate to contact me again with further details of thetype of negative control you require and we can investigate this further for you.

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