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    products/assay-kits/mitotox-complex-ii-iii-oxphos-activityassay-kit-ab109905.pdf

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Signal Transduction Metabolism Mitochondrial
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MitoTox™ Complex II + III OXPHOS Activity Assay Kit (ab109905)

  • Datasheet
  • SDS
  • Protocol Booklet
Submit a review Q&A (16)References (30)

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MitoTox™ Complex III OXPHOS Activity Assay (ab109905)
  • Mitochondrial ROS dependent functional deficiency and structural impairment in cardiac mitochondria after sepsis.

Key features and details

  • Assay type: Direct
  • Detection method: Colorimetric
  • Platform: Microplate reader
  • Assay time: 30 min

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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, Mammals
  • 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.

    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

  • 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 (III) 1 x 550µl
    Single-channel reagent reservoirs 2 units
  • Research areas

    • Signal Transduction
    • Metabolism
    • Mitochondrial
    • Kits/ Lysates/ Other
    • Kits
    • Cell Metabolism Kits
    • Other Metabolism Assay
    • Metabolism
    • Pathways and Processes
    • Mitochondrial Metabolism
    • Mitochondrial markers
  • 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

Images

  • MitoTox™ Complex III OXPHOS Activity Assay (ab109905)
    MitoTox™ Complex III OXPHOS Activity Assay (ab109905)

    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.

  • Mitochondrial ROS dependent functional deficiency and structural impairment in cardiac mitochondria after sepsis.
    Mitochondrial ROS dependent functional deficiency and structural impairment in cardiac mitochondria after sepsis.Image courtesy of Yao X et al.PLoS One. 2015; 10(10): e0139416. doi: 10.1371/journal.pone.0139416.

    Mitchondiral fractions from the heart tissue of Rats infected by S. pneumoniae, or given PBS sham control, were subjected to measurements of complex I-V activities. Complex I was measured with ab109721 (top left), Complex II + III were measured using ab109905 (top right), Complex IV was measured using ab109911 (bottom left) and Complex V was measured using ab109714 (bottom right).

    Freshly isolated mitochondrial pellets were resuspened in PBS supplemented with 10% detergent provided in the kits. Protein concentrations of these mitochondrial lysates were estimated and 25 μg (for complex I, IV and V) or 100 μg (for complex II+III) mitochondrial protein was used per reaction. Enzyme activities were measured spectrophotometricly in triplicate and expressed as changes of absorbance per minute per mg protein

Protocols

  • Protocol Booklet

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (30)

Publishing research using ab109905? Please let us know so that we can cite the reference in this datasheet.

ab109905 has been referenced in 30 publications.

  • Lei M  et al. Long non-coding RNA TUG1 sponges microRNA-9 to protect podocytes from high glucose-induced apoptosis and mitochondrial dysfunction via SIRT1 upregulation. Exp Ther Med 23:236 (2022). PubMed: 35222713
  • Kim M  et al. Evaluation of Parkin in the Regulation of Myocardial Mitochondria-Associated Membranes and Cardiomyopathy During Endotoxemia. Front Cell Dev Biol 10:796061 (2022). PubMed: 35265609
  • Han G  et al. Dihuang-Yinzi Alleviates Cognition Deficits via Targeting Energy-Related Metabolism in an Alzheimer Mouse Model as Demonstrated by Integration of Metabolomics and Network Pharmacology. Front Aging Neurosci 14:873929 (2022). PubMed: 35431901
  • Papachristodoulou A  et al. NKX3.1 Localization to Mitochondria Suppresses Prostate Cancer Initiation. Cancer Discov 11:2316-2333 (2021). PubMed: 33893149
  • Tsai ML  et al. IL-25 Induced ROS-Mediated M2 Macrophage Polarization via AMPK-Associated Mitophagy. Int J Mol Sci 23:N/A (2021). PubMed: 35008429
View all Publications for this product

Customer reviews and Q&As

Show All Reviews Q&A
Submit a review Submit a question

1-10 of 16 Abreviews or Q&A

Question

Will reagents in this kit allow me to measure complex activity in mouse mitochodria?

Read More

Abcam community

Verified customer

Asked on Jun 18 2013

Answer

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

Read More

Jeremy Kasanov

Abcam Scientific Support

Answered on Jun 18 2013

Question

Hello ! I want to measure the mitochondria complex III activity using a kit . I noted that there is a product titled MitoTox™ Complex III OXPHOS Activity Microplate Assay (ab109905). And i see that this product is designed to quick screening drugs. Can I use this kit to measure the complex III activity if i use my own sample mitochondria such as rat mitochondria extracted from heart instead of provided mitochondria in the kit and the other procedure is still the same? Can the antibody precoated onto the plate react with rat species ? And about measure other complex activity kit ,for example, for complex I activity kit ,there are two products. one is Complex I Enzyme Activity Microplate Assay Kit (ab109721) , the other is MitoTox™ Complex I OXPHOS Activity Microplate Assay (ab109903). I want to know what is the differences between this two kit. And what is the reason for there is only a kind of kit for complex III. why not have the activity kit for complex III like Complex I Enzyme Activity Microplate Assay Kit (ab109721) ? What is the difference between complex III and other complex ? Thank you very much. Urgently looking forward to your reply! Best regards! Qian L

Read More

Abcam community

Verified customer

Asked on Dec 28 2011

Answer

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 to identify a sample specific amount to use where the rate is acceptable. Fortunately, the kit is supplied with sufficient reagents to perform a test run to determine an acceptable rate. I hope this helps and if I can assist further, please do not hesitate to contact me.

Read More

Abcam Scientific Support

Answered on Dec 28 2011

Question

I am looking for Complex I-IV activity kits to study in vitro activity inhibitors after incubating mouse, rat, and human cells with the inhibitors prior to mitochondrial extraction. I am worried that either the kit will not detect the inhibited portion of the mechanism (such as ab109721 not being affected by rotenone since it detects the dehyrogenase activity and not the ubiquinone activity which is affected by rotenone) or the inhibitory affect will be reversed following mitochondrial extraction and sample preparation. Do you have any advice or specific product recommendations?

Read More

Abcam community

Verified customer

Asked on May 17 2014

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).

Read More

Kevin Hanson

Abcam Scientific Support

Answered on May 17 2014

Question

What is the difference between these 2 kits? Why does one immunocapture the complex II while the other does not? Are there any advantages/disadvantages of the immunocapture? Why is the complex III kit solely called complex III when based on the description it sounds like complex II activity is also measured.

Read More

Abcam community

Verified customer

Asked on Oct 23 2013

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.

Read More

Kevin Hanson

Abcam Scientific Support

Answered on Oct 23 2013

Question

Instrument can measure the absorbance at 490nm (not sure of range). Could this kit work with my instrument?

Read More

Abcam community

Verified customer

Asked on Oct 15 2013

Answer

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.

Read More

Caitlin Valued Customer

Abcam Scientific Support

Answered on Oct 15 2013

Question

I want to know whether I can use sodium azide as a complex IV inhibitor instead of KCN.

Read More

Abcam community

Verified customer

Asked on Sep 16 2013

Answer

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

Read More

Caitlin Valued Customer

Abcam Scientific Support

Answered on Sep 16 2013

Question

more details about sample preparation step:
- which lysis buffer to be used? RIPA, TX-100 or NP40 or no detergent at all?
- can samples be freeze-thawed
will use whole cell lysate (not a mito prep)

Read More

Abcam community

Verified customer

Asked on Jul 26 2013

Answer



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).


The kit requires a mitochondria preparation if the customer is not going to use it with the sample provided. It will not work on whole cells or whole cell extract. The customer must make a mito preparation from whole cells. They could use kit https://www.abcam.com/mitochondria-isolation-kit-for-cultured-cells-with-dounce-homogenizer-ab110171.html to make whole mitos from cells.


This assay is an in-solution assay (no antibody included in the kit) and therefore it can work with mitochondria preparations from any species.

Read More

Sybille Rex

Abcam Scientific Support

Answered on Jul 26 2013

Question

Inquiry: I have a question regarding ab109905. The detailed protocol booklet only covers the use of ab109905 for screening purposes using control bovine heart mitochondria. Can the kit method be adapted to use mitochondria isolated from animals treated with a potential complex III inhibitor? I have rat liver mitochondria which I would like to assay.... This work is likely to be published... in which case I would quote this product in print. If you could get back to me ASAP I would very much appreciate your assistance. Regards,

Read More

Abcam community

Verified customer

Asked on Mar 05 2013

Answer

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. The amount of mitochondria needed per test compound should also be optimized before testing the compounds.

The kit should work with any species, however the quality of the mitos will be very important.

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

Read More

Abcam Scientific Support

Answered on Mar 05 2013

Question

What we are interested in is whether the assays are sensitive enough for our application.


Will the abpromise mean that we are able to obtain a refund on the product if it is not sensitive enough?



Many Thanks

Read More

Abcam community

Verified customer

Asked on Aug 06 2012

Answer

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 uM)




1777.557
14157.28

3074.621
17427.79

2893.949
19235.95

2681.359
16799.75






ab109905

This assay was not designed to measure the activity of BHM, but rather the effect of a compound on a robust activity of BHM (which is provided with the kit). Ifyou want to usewhole mitochondria,the amount of mitochondria will have to be optimized, so that before you test compounds, you can measure a robust activity. The kit should work with any species, however the quality of the mitos will be very important.

Are you asking forthe sensitivity of a particular drug on the activity of complex III (II+III)? This of course is compound dependant, but we can provide the IC50 for some examples we have run in house.





Are you asking for the sensitivity of the assay at detecting BHM? The assay does not have an antibody. This is an in-solution assay which measures complex II+III in a 96 well plate. The assay was designed to use a robust signal of BHM so that compounds can be tested. Also, the assay does not have a “control protein complex II+III” to be able to produce the exact numbers I understand you wnat to see. We provide the BHM sample (which contains all the complexes of the electron transport chain). When customers run their assay, they should run the assay in the presence of compound and diluents. The diluent will typically be DMSO, which does not affect the assay at concentrations of 1 – 2%.





Do youwant to know the raw signal in mOD/min in the presence of DMSO?. We can provide this as a reference guide, but not for ab109905, because the raw number is dependent on the BHM batch preparation and the substrates batch preparation. We can however giveout raw data from the QC of a particular batch, which is performed without DMSO.



I hope this information is helpful and wish you good luck with your research.

Read More

Abcam Scientific Support

Answered on Aug 06 2012

Question

i bought the kit as "ab109905" from your company and i assayed the inhibition effect of desire compound on the complex III activity via dose response assay.Im going toalculate the complex III activity but i faced a problem!whats the background rate (0.3 mOD/min) that has writenin the protocole?

please explain the calculation section for me step by step!

Read More

Abcam community

Verified customer

Asked on May 21 2012

Answer

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 average of your sample readings for a particular treatment, and subtracting the background rate. This activity measurement can then be used to compare different methods of treatment.

I hope this helps, if you have any further questions, please let me know and I will be happy to help you further.

Read More

Abcam Scientific Support

Answered on May 21 2012

1-10 of 16 Abreviews or Q&A

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