MitoTox™ Complete OXPHOS Activity Assay Kit (5 Assays) (ab110419)

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MitoTox™ Complete OXPHOS Activity Assay Kit (5 Assays) (ab110419)
  • MitoTox™ Complete OXPHOS Activity Assay Kit (5 Assays) (ab110419)
  • MitoTox™ Complete OXPHOS Activity Assay Kit (5 Assays) (ab110419)
  • MitoTox™ Complete OXPHOS Activity Assay Kit (5 Assays) (ab110419)
  • MitoTox™ Complete OXPHOS Activity Assay Kit (5 Assays) (ab110419)
  • MitoTox™ Complete OXPHOS Activity Assay Kit (5 Assays) (ab110419)

Key features and details

  • Assay type: Quantitative
  • Detection method: Colorimetric
  • Platform: Microplate reader
  • Sample type: Inhibitor compounds

Overview

  • Product name

    MitoTox™ Complete OXPHOS Activity Assay Kit (5 Assays)

  • Detection method

    Colorimetric

  • Sample type

    Inhibitor compounds

  • Assay type

    Quantitative

  • Species reactivity

    Reacts with: Cow, Human

  • Product overview

    MitoTox™ Complete OXPHOS Activity Assay Kit (5 assays) (ab110419) is designed for testing the direct inhibitory effect of compounds on the 5 OXPHOS complexes. Inhibitory effects of compounds on OXPHOS Complexes 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 specific Complex activity can be tested at 11 different data points in triplicate.


    Complexes I, II, IV and V activity are each immunocaptured from bovine heart mitochondria by specific antibodies on 96-well microplates, and their activities measured by simple spectrophotometric assays. Complex III activity is measured by an in-solution colorimetric assay.


    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

    This kit consists of the following assays:

    MitoTox™ Complex I OXPHOS Activity Assay Kit (ab109903)

    MitoTox™ Complex II OXPHOS Activity Assay Kit (ab109904)

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

    MitoTox™ Complex IV OXPHOS Activity Assay Kit (ab109906)

    MitoTox™ Complex V OXPHOS Activity Assay Kit (ab109907)

     

    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 5 x 96 tests
    Detergent 1 x 1ml
    12-channel reagent reservoirs 10 units
    1X Blocking Buffer 1 x 70ml
    1X Complex III Mito Buffer 1 x 1ml
    1X Complex IV Mito Buffer 1 x 110ml
    1X Mito Buffer 2 x 5ml
    1X Succinate Solution 1 x 12ml
    20X Buffer 1 x 5ml
    20X Wash Buffer 2 x 5ml
    96-well microplate 1 unit
    Bovine heart mitochondria 4 x 360µl
    Bovine Heart Mitochondria 1 x 90µl
    Bovine heart mitochondria (5 mg/mL) 1 x 300µl
    Complex I Activity Buffer 1 x 24ml
    Complex II Activity Buffer 1 x 24ml
    Complex V Activity Buffer 1 x 24ml
    Cytochrome c (III) 1 x 550µl
    DCPIP/DCIP 1 x 250µl
    Detergent 1 x 20µl
    Detergent 2 x 100µl
    Blocking Powder 1 x 1.8g
    Phospholipids 2 x 6ml
    Pre-coated 96-well microplate 4 units
    Reagent C (Reduced Cytochrome c) 2 x 1ml
    Single-channel reagent reservoirs 2 units
    Succinate 1 x 500µl
    Ubiquinone 1 1 x 60µl
    Ubiquinone 2 1 x 60µl

  • Research areas

Images

  • MitoTox™ Complete OXPHOS Activity Assay Kit (5 Assays) (ab110419)
    MitoTox™ Complete OXPHOS Activity Assay Kit (5 Assays) (ab110419)

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

  • MitoTox™ Complete OXPHOS Activity Assay Kit (5 Assays) (ab110419)
    MitoTox™ Complete OXPHOS Activity Assay Kit (5 Assays) (ab110419)

    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.

  • MitoTox™ Complete OXPHOS Activity Assay Kit (5 Assays) (ab110419)
    MitoTox™ Complete OXPHOS Activity Assay Kit (5 Assays) (ab110419)

    Typical dose response curve for KCN. Assay was performed following the Dose Response Assay Procedure using KCN, a well known Complex IV inhibitor. KCN was prepared in 0.1 M NaOH to generate a 100 mM stock. Starting with a 100 µM final concentration in well, 1:10 serial dilutions of KCN were generated.

  • MitoTox™ Complete OXPHOS Activity Assay Kit (5 Assays) (ab110419)
    MitoTox™ Complete OXPHOS Activity Assay Kit (5 Assays) (ab110419)

    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.

  • MitoTox™ Complete OXPHOS Activity Assay Kit (5 Assays) (ab110419)
    MitoTox™ Complete OXPHOS Activity Assay Kit (5 Assays) (ab110419)

    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.

  • MitoTox™ Complete OXPHOS Activity Assay Kit (5 Assays) (ab110419)
    MitoTox™ Complete OXPHOS Activity Assay Kit (5 Assays) (ab110419)

    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.

References (7)

Publishing research using ab110419? Please let us know so that we can cite the reference in this datasheet.

ab110419 has been referenced in 7 publications.

  • Nomiyama K  et al. Targeted metabolome analysis of the dog brain exposed to PCBs suggests inhibition of oxidative phosphorylation by hydroxylated PCBs. Toxicol Appl Pharmacol N/A:114620 (2019). PubMed: 31195005
  • Yi X  et al. SIRT3-Dependent Mitochondrial Dynamics Remodeling Contributes to Oxidative Stress-Induced Melanocyte Degeneration in Vitiligo. Theranostics 9:1614-1633 (2019). PubMed: 31037127
  • Xuan J  et al. Mitochondrial dysfunction induced by leflunomide and its active metabolite. Toxicology 396-397:33-45 (2018). PubMed: 29427785
  • Zhu H  et al. MiR-138 protects cardiac cells against hypoxia through modulation of glucose metabolism by targetting pyruvate dehydrogenase kinase 1. Biosci Rep 37:N/A (2017). PubMed: 28899927
  • Zhang C  et al. Sorafenib targets the mitochondrial electron transport chain complexes and ATP synthase to activate the PINK1-Parkin pathway and modulate cellular drug response. J Biol Chem 292:15105-15120 (2017). PubMed: 28673964
  • Nguyen KC  et al. Mitochondrial Toxicity of Cadmium Telluride Quantum Dot Nanoparticles in Mammalian Hepatocytes. Toxicol Sci 146:31-42 (2015). PubMed: 25809595
  • Lai K  et al. Integrated Compound Profiling Screens Identify the Mitochondrial Electron Transport Chain as the Molecular Target of the Natural Products Manassantin, Sesquicillin, and Arctigenin. ACS Chem Biol N/A:N/A (2012). PubMed: 23138533

Customer reviews and Q&As

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Screening the Effect of 10 Different Chemicals on Different Mitochondrial Electron Transport Chain Complexes ( CI - CV)

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Actually, this kit helped me to check the effect of 10 different chemicals on the 5 mitochondrial electron transport chain complexes. I had some queries regarding the design of some assays and I contacted the Abcam support team. They really helped me and provided me with good technical support.

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Verified customer

Submitted Nov 28 2019

Question

As we are looking for the assay to evaluate mitochondrial toxicity of our chemicals for human. We have queries regarding your kit "MitoTox™ Complete OXPHOS Activity Assay Kit​" (5 Assays) ab110419, as mentioned in your kit protocol

Human relevance of the kit

Sample and sample preparation in case of human

Species reactivity said only for bovine not for human

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Answer

On our datasheet we state that this kit can be used with human and cow.

Therefore yes I can confirm that you can use it on your human samples. What I think may be confusing is that we supply bovine heart mitochondria rather than human.

To isolate mitochondria from human PBMCs I suggest you use the Mitochondria Isolation Kit for Cultured Cells (ab110170) (https://www.abcam.com/ab110170) and start with a minimum of 8x10^7 PBMC cells for a yield of 1mg. The mitochondria from PBMCs may be frozen prior to detergent extraction. The higher the mitochondrial preparation protein concentration, the longer the stability at -80C storage. The standard mitotox kits are shipped with frozen whole bovine heart mitochondria at 5.5mg/mL.

I suggest you do side by side detergent extraction of the bovine heart mitochondria with the PMBC mitochondria and use the BHM mitos in at least 2 or 3 wells of the plate to ensure that the assay is working.

I do not recommend using this kit with whole blood.

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Question

The dose response curves of our two compounds from the first experiment are good, but those from the second experiment are weird.

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Answer


It is crucial that this type of data is analyzed based on the actual kinetic trace (via softmax software). When a reduction trace is superimposed you will notice that the kinetic change in signal is not constant for the entire assay. The speed of change will vary over the course of the read. Activity assays should be measured in the region where the maximum drop occurs. Most software is capable of adding a lag and an end phase to the analysis. When this is applied, maximum mOD/min is calculated by the software. This allows to have greater reproducibility.

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Question

Does this assay work on frozen mitochondria? Will it work with human PBMC isolated mitochondria?

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Answer

The mitotox kits will work with mitochondria from any human tissue. Therefore it will work with mitochondria from human PBMCs.


To isolate mitochondria from human PBMCs I suggest the customer to use ab110170 (Mitochondria Isolation Kit for Cultured Cells) and start with a minimum of 8x10^7 PBMC cells for a yield of 1mg. The mitochondria from PBMCs may be frozen prior to detergent extraction. The higher the mitochondrial preparation protein concentration, the longer the stability at -80ºC storage. The standard mitotox kits are shipped with frozen whole bovine heart mitochondria at 5.5mg/mL. I suggest the customer to do side by side detergent extraction of the bovine heart mitochondria with the PMBC mitochondria and used the BHM mitos in at least 2 or 3 wells of the plate to ensure that the assay is working.


I do not recommend using the MitoTox kits with whole blood.

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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

Hi again,

Thanks for the info. Please can you give me contact info for MitoSciences? I’d like to talk directly to them about working on a 384-well format.

Cheers

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Answer

Thank you for contacting Abcam.

MitoSciences is part of the Abcam family now, and I have directly contacted them regarding your inquiry. They responded that the 384 well plate format has not been validated or produced. Since Abcam and MitoSciences are one in the same, they will let me know if anything changes and they are able to produce ab110419 in 384-well plate format. While this is not currently being explored I will inform you if anything changes.

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

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

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Question

Please can you find out from the folks at MitoSciences if they have successfully formatted the OXPHOS ab110419 assays at 384-well? I do not want to place an order on a format that has not been validated.

Cheers,

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Answer

Thank you for contacting Abcam.

I contacted the supplier, and they responded that ab110419 has not been validated in 384 well plate format.

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

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

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Question

Product code: 110419

Inquiry: Hi guys, I was wondering if you have the MitoTox™ Complete OXPHOS Activity Assay Kits available in 384-well format?

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Answer

Thank you for contacting Abcam.

We only make custom products for bulk orders of 10 or more products. If you would like to inquire about placing a bulk order for ab110419 in 384-well format please reply to this email, and we will see if the supplier can produce the kit in the format you desire. If the supplier will produce ab110419 in 384-well format, we will send you back an invoice specific for your custom bulk order.

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

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link: https://www.abcam.com/index.html?pageconfig=resource&rid=15447

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Question


Inquiry: Hi, We are interested in testing electron transport chain enzyme activity on frozen human liver samples. Is this the appropriate kit- MitoTox™ Complete OXPHOS Activity Assay Kit (5 Assays) (ab110419) ? Thanks

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Answer

Thank you for contacting us.
The assays measure the direct effects of drugs on the 5 OXPHOS complexes and can be used on human samples.

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

Use our products? Submit an Abreview. Earn rewards!
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Question

Hello,

Ideally, I was looking for something to elucidate the differences (oxphos)on normal human brain and human brain tumours with and without cytotoxic compounds. Is this achievable? Any advice would be gratefully appreciated.

Kind regards,

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Answer

I now have further information to share with you.

The antibodies used in the MitoTox™ Complete OXPHOS Activity Assay Kit (5 Assays), ab110419,to coat the plates are cross-reactive againstthe human proteins. The kit can therefore be used with Human mitochondria, however the signal will be better with bovine heart mitochondria.

My colleague at Mistosciences has suggested that you could test the compounds in vitro with the bovine mitochondria provided as well as with mitochondria from normal human brain and mitochondria from tumor brain.The bovine samples will allowyou to ensure that the kit works as expected.It will also giveyou a reference point in regards to whether the compound inhibit the enzyme or not.
The Mitochondria may be prepared with kit https://www.abcam.com/Mitochondria-Isolation-Kit-for-Tissue-ab110168.html. For brain samples, the protocol calls for 0.3 – 0.4g of tissue to expect a yield of 4 – 5mg. Following the protocol, the mitochondria may be frozen at -80°C as whole mitochondria and can be solubilized on the day of the assay side by side with the bovine heart mitochondria provided.
Another option is to use the regular activity kits, however those kits are not optimized to be used with compounds as they do not provide null wells. These null wells are important as some compounds can react with the components of the activity buffer in the absence of enzyme.
https://www.abcam.com/Complex-I-Enzyme-Activity-Microplate-Assay-Kit-ab109721.html
https://www.abcam.com/Complex-II-Enzyme-Activity-Microplate-Assay-Kit-ab109908.html
https://www.abcam.com/ATP-synthase-Enzyme-Activity-Microplate-Assay-Kit-ab109714.html
https://www.abcam.com/Complex-IV-Human-Enzyme-Activity-Microplate-Assay-Kit-ab109909.html



I hope this information has been of help. If you have any further questions, please do not hesitate to contact us again.

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