Overview

  • Product name

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

    Colorimetric
  • Assay type

    Direct
  • Assay time

    4h 00m
  • Species reactivity

    Reacts with: Mouse, Cow, Human
  • Product overview

    MitoTox™ Complex II OXPHOS Activity Assay Kit (ab109904) is designed for testing the direct inhibitory effect of compounds on Complex II activity in only 4 hours. Complex II extracted from the provided bovine heart mitochondria (a rich source of Complex II) is immunocaptured by specific antibodies on the plate. Complex II activity can be observed as decrease in absorbance at OD 600 nm. The intra-assay and inter-assay variation of this assay are both <15%.


    Inhibitory effects of compounds on Complex II 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 II 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

    Please store Succinate, Ubiquinone 2, Bovine Heart Mitochondria, DCPIP at -80°C and 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

  • Typical dose response curve for TTFA (2-thenoyltrifluoracetone). Assay was performed following the Dose Response Assay Procedure using TTFA, a well known Complex II inhibitor. TTFA was prepared in DMSO to generate a 100 mM stock. Starting with a 500 µM final concentration in well, 1:2 serial dilutions of TTFA were generated.

Protocols

References

This product has been referenced in:

  • Subedi A  et al. A novel inhibitor of tumorspheres reveals the activation of the serine biosynthetic pathway upon mitochondrial inhibition. FEBS Lett 593:763-776 (2019). Read more (PubMed: 30874300) »
  • Landry GM  et al. Diglycolic acid inhibits succinate dehydrogenase activity in human proximal tubule cells leading to mitochondrial dysfunction and cell death. Toxicol Lett 221:176-84 (2013). Read more (PubMed: 23827505) »
See all 3 Publications for this product

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

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

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 enzyme.

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Question
Answer

Thank you for contacting us.

The 10x detergent in the kit is 10% Lauryl maltoside, which can also be purchased separately as ab109857.

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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https://www.abcam.com/abreviews

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Answer

Thank you for your call and for your patience while I've been in touch with the lab about ab109904.

My contact at the lab gave the following reply: "Yes, a pH of 2 will destroy the assay. You will need to bring the pH to neutral ( 7 – 7.4) to test in the assay, and thereforeyou will have to test the salt version of the acid and not the acid directly in the assay. For example = Malonic acid is a known inhibitor of complex II (as it has been shown in the literature multiple times). We have tested in the past the effect of Potassium and Sodium Malonate (which is Malonic acid after pH has been adjusted with NaOH or KOH) and have found that Malonate (in the salt form) inhibits the activity of Complex II when tested with the MTOX2 assay."

I hope this information is useful, but please let me know if you have any further questions and I'll be happy to help. Have a great day!

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