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

製品の概要

  • 製品名MitoTox™ Complex II +  III OXPHOS Activity Microplate Assay
    Complex III キット 製品一覧
  • 検出方法Colorimetric
  • テスト
    1 x 96 well plate
  • アッセイタイプDirect
  • 全工程の試験時間
    0h 30m
  • ステップMultiple steps standard assay
  • 種交差性
    交差種: Cow, A wide range of mammals
  • 製品の概要

    The MitoTox™ OXPHOS Complex III Activity Kit is designed for screening the direct inhibitory effects of compounds on Complex II and III activity. Bovine heart mitochondria are provided in this kit as they are a rich source of these two enzymes. The kit contains succinate (Solution 1) which is the electron donor of Complex II, as well as cytochrome c (oxidized), the electron acceptor of Complex III. Ubiquinone, the electron acceptor of Complex II, is present in the mitochondria. The rate of the coupled Complex II+III reaction is measured by monitoring the conversion of cytochrome c in its oxidized form, cytochrome c (oxidized), to its reduced form, cytochrome c (reduced), as a linear increase in absorbance at exactly  550 nm (reading the absorbance at 540 nm will not be sufficient for this assay). The kit requires the use of rotenone and KCN (not supplied with the kit). Addition of rotenone, an inhibitor of Complex I, ensures that all reduction of Cytochrome c is via Complex II. Addition of KCN ensures that there is no re-oxidation of Cytochrome c by Complex IV. The user may add antimycin, an inhibitor of Complex III activity, in the screening procedure as a positive control. 50% inhibition of Complex III activity is obtained with 22 ± 4 nM antimycin at assay conditions described in the protocol.

     

  • 特記事項

    Store at -80°C: Bovine Heart Mitochondria, Cytochrome c (oxidized).

    Store at 4°C: Succinate Solution, Microplate, Mito Buffer and Reagent Reservoirs: Reagent Reservoirs may be stored at room temperature.

  • アプリケーション適用あり: Functional Studiesmore details
  • 試験プラットフォームMicroplate

製品の特性

  • 関連性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.
  • 別名
    • coenzyme Q : cytochrome c — oxidoreductase
    • cytochrome bc1 complex

アプリケーション

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.

アプリケーション Abreviews 特記事項
Functional Studies Use at an assay dependent dilution.

MitoTox™ Complex II +  III OXPHOS Activity Microplate Assay 画像

プロトコール

MitoTox™ Complex II +  III OXPHOS Activity Microplate Assay (ab109905) 使用論文

This product has been referenced in:
  • 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) »
  • Yao X  et al. Estrogen-provided cardiac protection following burn trauma is mediated through a reduction in mitochondria-derived DAMPs. Am J Physiol Heart Circ Physiol 306:H882-94 (2014). Rat . Read more (PubMed: 24464748) »

See all 5 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"