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

概述

  • 产品名称MitoTox™ Complex II + III OXPHOS Activity Assay试剂盒
    参阅全部 Complex III 试剂盒
  • 检测方法Colorimetric
  • 检测类型Direct
  • 检测时间
    0h 30m
  • 种属反应性
    与反应: Cow, Mammal
  • 产品概述

    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. 


     

  • 说明

    Store Bovine Heart Mitochondria and Cytochrome c (oxidized) at -80°C. Store all other components at 4°C.

  • 经测试应用适用于: Functional Studiesmore details
  • 平台Microplate

性能

  • 相关性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.
  • 别名
    • coenzyme Q : cytochrome c — oxidoreductase
    • cytochrome bc1 complex

应用

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.

应用 Ab评论 说明
Functional Studies Use at an assay dependent dilution.

MitoTox™ Complex II + III OXPHOS Activity Assay Kit 图像

  • 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.

实验方案

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

This product has been referenced in:
  • Seo JH  et al. The Mitochondrial Unfoldase-Peptidase Complex ClpXP Controls Bioenergetics Stress and Metastasis. PLoS Biol 14:e1002507 (2016). Read more (PubMed: 27389535) »
  • Yao X  et al. Mitochondrial ROS Induces Cardiac Inflammation via a Pathway through mtDNA Damage in a Pneumonia-Related Sepsis Model. PLoS One 10:e0139416 (2015). Functional Studies ; Rat . Read more (PubMed: 26448624) »

See all 6 Publications for this product

Product Wall

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