MitoTox™ Complex II + III OXPHOS Activity Assay Kit (ab109905)

Overview

  • Product name
    MitoTox™ Complex II + III OXPHOS Activity Assay Kit
    See all Complex III kits
  • Detection method
    Colorimetric
  • Assay type
    Direct
  • Assay time
    0h 30m
  • Species reactivity
    Reacts with: Cow, Mammal
  • Product overview

    MitoTox™ Complex II+ III OXPHOS Activity Assay Kit (ab109905) is designed for testing the direct inhibitory effect of compounds on Complex III activity in only 30 minutes. The assay is performed using whole bovine heart mitochondria, a rich source of OXPHOS complex. Succinate (electron donor of Complex II) and oxidized cytochrome c (electron acceptor of Complex III) are added to the mitochondria to start the electron transfer pathway that takes place during oxidative phosphorylation. The rate of couple Complex II + III reaction is measured by monitoring the conversion of oxidized cytochrome c into reduced form, which can be observed as increase in absorbance at OD 550 nm. The assay requires rotenone and KCN (not supplied): rotenone (Complex I inhibitor) blocks electron transfer to ubiquinone, ensuring that all reduction of cytochrome c happens via Complex II. The addition of KCN (Complex IV inhibitor) ensures that there is no re-oxidation of cytochrome c. The intra-assay and inter-assay variation of this assay are both < 10%.


    Inhibitory effects of compounds on Complex III 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 III 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 Bovine Heart Mitochondria and Cytochrome c (oxidized) at -80°C. Store all other components at 4°C.

  • Tested applications
    Suitable for: Functional Studiesmore details
  • Platform
    Microplate

Properties

  • Storage instructions
    Please refer to protocols.
  • Components 96 tests
    12-channel reagent reservoirs 2 units
    1X Complex III Mito Buffer 1 x 1ml
    1X Succinate Solution 1 x 12ml
    96-well microplate 1 unit
    Bovine heart mitochondria (5 mg/mL) 1 x 300µl
    Cytochrome c (oxidized) 1 x 550µl
    Single-channel reagent reservoirs 2 units
  • Research areas
  • Relevance
    Complex II (succinate-ubiquinone oxidoreductase), one of the mitochondrial respiratory chain complexes, transfers electrons from succinate generated during the citric acid cycle to Complex III (ubiquinolcytochrome c oxidoreductase), via a mobile electron shuttle, ubiquinone. Complex III transfers electrons to Complex IV (cytochrome c oxidase) via another mobile electron shuttle, cytochrome c.
  • Alternative names
    • coenzyme Q : cytochrome c — oxidoreductase
    • cytochrome bc1 complex

Applications

Our Abpromise guarantee covers the use of ab109905 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
Functional Studies Use at an assay dependent dilution.

Images

  • Typical dose response curve for antimycin A. Assay was performed following the Dose Response Assay Procedure using antimycin A, a well known Complex III inhibitor. Antimycin A was prepared in DMSO to generate a 10 mM stock. Starting with a 150 µM final concentration in well, 1:4 serial dilutions of antimycin A were generated.

Protocols

References

This product has been referenced in:
  • Kon N  et al. A small-molecule DS44170716 inhibits Ca(2+)-induced mitochondrial permeability transition. Sci Rep 7:3864 (2017). Read more (PubMed: 28634393) »
  • Seo JH  et al. The Mitochondrial Unfoldase-Peptidase Complex ClpXP Controls Bioenergetics Stress and Metastasis. PLoS Biol 14:e1002507 (2016). Read more (PubMed: 27389535) »

See all 7 Publications for this product

Customer reviews and Q&As

Yes. This assay is an in-solution assay (not immunocapture based) and therefore it would work on whole mitochondria from any species.

Thank you for your enquiry and your interest in our products. ab109905 is the only assay which is not an immunoassay. The supplied bovine sample can be replaced with a mitochondrial sample from any other species. This may require some optimization ...

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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
http://www.abcam.com/mitotox%E2%84%A2-complex-i...

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ab109904 measures solely complex II, whereas ab109905 measures complexes II+III.
We do not offer an immunocapture activity assay for Complex III only. The reason is that we have not generated an antibody capable of immunocapturing the native enz...

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Cytochrome C should only be measured at 550 nm using this kit. the peak spectra of reduced cytochrome c is at 550 nm. The wavelength can't vary from this, unfortunately, so 490 nm and other wavelengths will not be sufficient to use with the kit.

You can use azide instead of KCN, and 1 mM azide should inhibit 50% of COX activity.



The MitoTox complex III oxphos assay does not require a detergent. The whole bovine mitochondria provided with the kit must be run according to the protocol (no further preparation required, simply dilute in the buffer provided).

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Thank you for contacting us.

This assay is designed to measure the effect of a compound on a robust activity of BHM (which is provided with the kit). In case of other whole mitochondrial samples the user should optimize the assay themselves. T...

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Thank you for your reply.

The only data for sensitivity we have at the moment for ab112150 can be found on the datasheet as graph. I also include the actual numbers here:

NIR fluorescence, four replicates.

Control FCCP (20...

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Thank you for contacting us.

The 0.3 mOD/min is an example figure, your results may vary slightly. This background rate is the rate observed in your control wells that do not contain any mitochondria. The activity is measured by taking the av...

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1-10 of 16 Abreviews or Q&A

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

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