MitoTox™ Complex I OXPHOS Activity Assay Kit (ab109903)

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Overview

  • Product name

    MitoTox™ Complex I OXPHOS Activity Assay Kit
    See all Complex I kits

  • Detection method

    Colorimetric

  • Sample type

    Inhibitor compounds

  • Assay type

    Quantitative

  • Assay time

    5h 00m

  • Product overview

    MitoTox™ Complex I OXPHOS Activity Assay (ab109903) is designed for testing the direct inhibitory effect of compounds on Complex I activity in only 5 hours. Complex I extracted from the provided bovine heart mitochondria (a rich source of Complex I) is immunocaptured by specific antibodies on the plate. Complex I 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 I 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, Ubiquinone 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

  • MitoTox™ Complex I OXPHOS Activity Assay (ab109903)
    MitoTox™ Complex I OXPHOS Activity Assay (ab109903)

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

Protocols

References

This product has been referenced in:

  • Thomas LW  et al. CHCHD4 confers metabolic vulnerabilities to tumour cells through its control of the mitochondrial respiratory chain. Cancer Metab 7:2 (2019). Read more (PubMed: 30886710) »
  • Cameron AR  et al. Metformin selectively targets redox control of complex I energy transduction. Redox Biol 14:187-197 (2018). Read more (PubMed: 28942196) »
See all 7 Publications for this product

Publishing research using ab109903? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-10 of 17 Abreviews or Q&A

Question


I am really interested in buying this kit (MitoTox™ Complex I OXPHOS
Activity Microplate Assay (ab109903)). But I need to know if the whole
pre-coated 96-well microplate must be used in only one assay. In other
words, Can I re-use this microplate in a second assay that will be
done in a different day?

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Answer





The plate is one solid plate and not removable strips. Plate is best used at once as there is risk to unused wells getting wet, etc. We have not tried this and therefore cannot guarantee, but you could try putting a plate seal over unused wells to save them for a second experiment.

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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?

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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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Question

I have used this kit before and have never had any problems. My data this particular kit shows that the 340 nm absorbance decreases over time in ALL wells. The absorbance should decrease over time in rows B-G but not in row A and row H, which are supposed to be blanks.

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Answer

Thank you for contacting us.

I have checked with our MitoSciences lab and they sent the attached QC data for the plates used in that lot. Additionally, these kits are designed to be run with developmental compounds. On occasion, we have seen that some developmental compounds have redox activity on their own (they react with the components of the kit) in the absence of sample. In this scenario, you will see activity in the blank wells and this activity should be subtracted from the activity in the test wells. Was your plate run with or without compounds?

If you would like to receive a replacement kit, we do have kits in stock with plates from a different lot. If you have purchased the kit in the past six months I would be happy to send you a free of charge replacement. Could you please let me know your original order or PO number? I will be happy to help you further once I have this additional information.

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Question

Pour faire suite à mes problème technique avec le kit de complex I.
J'ai refait une manip avec rotenonne préparé fraichement et un de nos aliquots conservé à -20°C. Il n'y a pas de différence importante entre les 2.
J'ai tester de faire l'expérience avec 60 ul de phospholipides au lieu de 40ul comme indiqué et là mes résultats sont bien différent et j'obtiens du coup une IC50 pour la rotenone de l'ordre de 38 nM alors que la première manip j'avais plus de 400nM en utilisant que 40ul.
Auriez vous une explication, est ce que les phospholipides serait pas un facteur limitant?

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Answer

Les phospholipides peuvent en effet être le paramètre critique pour le bon fonctionnement du kit MTOX I.Ils ne sont pas stables si ils sont conservés à 4ºC et doivent être impérativement stockés à -20 ou -80ºC.

Le kit ab109903 devrait vous avoir été livré avec de la carboglace, pourriez-vous s'il vous plait me confirmer ceci?

J'espère que ces informations vous seront utiles. N'hésitez pas à nous contacter de nouveau si vous avez d'autres questions.

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Question

Bonjour,

Pour faire suite à mes problème technique avec le kit de complex I.
J'ai refait une manip avec rotenonne préparé fraichement et un de nos aliquots conservé à -20°C. Il n'y a pas de différence importante entre les 2.
J'ai tester de faire l'expérience avec 60 ul de phospholipides au lieu de 40ul comme indiqué et là mes résultats sont bien différent et j'obtiens du coup une IC50 pour la rotenone de l'ordre de 38 nM alors que la première manip j'avais plus de 400nM en utilisant que 40ul.
Auriez vous une explication, est ce que les phospholipides serait pas un facteur limitant?

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Answer

Je suis ravi que les suggestions aient été utiles.

J'ai transmis votre question au laboratoire et vous contacterai de nouveau dès que j'aurai reçu une réponse.

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Question

Le kit et neuf est bien entreposé. Par contre notre roténone est conservé en aliquote à -20

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Answer

Merci de votre réponse.

Le laboratoire vient de m'indiquer que la valeur de laDO initiale mesurée lors des tests de contrôle qualité était de 1 pour le lot GR78475-2.

Le problème rencontré provient doncprobablement dela dégradation de la roténone.

Assurez-vous également que les mesures sont effectuées à la bonne longueur d'onde.

Merci de m'indiquer si les recommandations ont permis d'améliorer les résultats.

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Question

Product code: 109903
Lot number: GR78475-2
Inquiry: Bonjour, J'ai testé votre kit MitoTox complex I avec une dose réponse de rotenone. Vous stipulé que l'IC50 devrait etre vers 13 nM. Or d'apres mon test je suis plutôt a 413nM. Aussi ma DO au départ n'est pas très élevé tourne autour de 0.5. Dois je avoir une DO au départ plus élevé ou est ce correcte. Qu'est ce qui pourrait expliqué que mon IC50 soit bien plus élevé que ce que vous indiqué pour la rotenone.

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Answer

Merci de nous avoir contactés.

La DO initiale (0.5) est en effet un peu faible et pourrait expliquer la valeur anormalementélevée de l'IC50.

Une des raisons expliquant la faible DO est l'age du kit et les conditions de stockage. Deux températures sont à appliquer à ce kit. Le tampon de réaction doit être stocké à -80ºC pour préserver le NADH sensible à de plus hautes températures.

Pourriez-vous me confirmer les conditions de stockage du kit et de ce tampon en particulier?

Une autre raison pour expliquer l'IC50 élevé est l'échantillon lui-même. Au laboratoire, le kit est testé avec de la rotenone (de chez Sigma)fraîchement diluée dans le DMSO. Une conservation à -20 ou -80ºC de la solution de rotenone dans le DMSO n'est pas conseillée.

J'espère que ces informations vous seront utiles. N'hésitez pas à nous contacter de nouveau si vous avez d'autres questions.

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Question

What is the solvent for the Ubiquinone 1 component?

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Answer

Thank you for your enquiry.

The solvent for the Ubiquinone 1 component is 100% ethanol.

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Question

Can I confirm the following things about this Complex I Immunocapture Kit?

1) This product only contains antibodies crosslinked to agarose beads, and nothing else. The name "Immunocapture Kit" was confusing as I thought there were other components in the kit.

2) In the product overview, [Thus the enzyme retains NADH-ferricyanide, NADH-CoQ1 and NADH hexaaminoruthenium reductase activities after isolation, and with added lipids it also shows significant rotenone sensitivity] , there is a mention about adding lipids to show rotenone sensitivity. Do you have a recommend lipid product, and protocol for this to be done?

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Answer

Thank you for your enquiry. Please find the answers to your questions below.

1) Thank you for your feedback. We will take this into consideration with the production team.

2) Typically customers have used this kit in combination with Complex I activity assays published in the literature. However if you would like to detect rotenone sensitivity, then this ab109903 MitoTox™ Complex I OXPHOS Activity Microplate Assay would be more appropriate. It is a plate based assay that contains lipids and is rotenone sensitive. However, the lipid content of this kit is proprietary.

I have attached a paper showing how to perform activity assay after immunocapture with the beads. This protocol uses BSA instead of the lipid (see figure 2 under results).

A Functionally Active Human F1F0 ATPase Can be Purified by Immunocapture from Heart Tissue and Fibroblast Cell Lines. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 37, pp. 33906–33912, 2002

I hope this information will be helpful.

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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

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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.

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