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MitoTox™ Complex IV OXPHOS Activity Assay Kit (ab109906)

Submit a review Q&A (2)References (7)
MitoTox™ Complex IV OXPHOS Activity Assay (ab109906)

    Key features and details

    • Assay type: Direct
    • Detection method: Colorimetric
    • Platform: Microplate reader
    • Assay time: 5 hr

    製品の概要

    • 製品名

      MitoTox™ Complex IV OXPHOS Activity Assay Kit
      Complex IV キット 製品一覧

    • 検出方法

      Colorimetric

    • アッセイタイプ

      Direct

    • 全工程の試験時間

      5h 00m

    • 種交差性

      交差種: Cow, Human

    • 製品の概要

      MitoTox™ Complex IV OXPHOS Activity Assay Kit (ab109906) is designed for testing the direct inhibitory effect of compounds on Complex IV activity in only 5 hours. Complex IV extracted from the provided bovine heart mitochondria (a rich source of Complex IV) is immunocaptured by specific antibodies on the plate. Complex IV activity can be observed as decrease in absorbance at OD 550 nm. The intra-assay and inter-assay variation of this assay are both <10%.


      Inhibitory effects of compounds on Complex IV 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 IV 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. 


       


       

    • 特記事項

      Store Bovine Heart Mitochondria and Reagent C 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.

    • 試験プラットフォーム

      Microplate reader

    製品の特性

    画像

    • MitoTox™ Complex IV OXPHOS Activity Assay (ab109906)
      MitoTox™ Complex IV OXPHOS Activity Assay (ab109906)

      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.

    参考文献 (7)

    ab109906 を使用した論文を発表された方は、こちらまでお知らせください。データシートに掲載させていただきます。

    ab109906 は 7 報の論文で使用されています。

    • Ma Y  et al. IRX3 plays an important role in the pathogenesis of metabolic-associated fatty liver disease by regulating hepatic lipid metabolism. Front Endocrinol (Lausanne) 13:895593 (2022). PubMed: 35957832
    • Papachristodoulou A  et al. NKX3.1 Localization to Mitochondria Suppresses Prostate Cancer Initiation. Cancer Discov 11:2316-2333 (2021). PubMed: 33893149
    • Kakoki M  et al. Cyanocobalamin prevents cardiomyopathy in type 1 diabetes by modulating oxidative stress and DNMT-SOCS1/3-IGF-1 signaling. Commun Biol 4:775 (2021). PubMed: 34163008
    • Baccelli I  et al. Mubritinib Targets the Electron Transport Chain Complex I and Reveals the Landscape of OXPHOS Dependency in Acute Myeloid Leukemia. Cancer Cell 36:84-99.e8 (2019). PubMed: 31287994
    • Wang Y  et al. Metformin Improves Mitochondrial Respiratory Activity through Activation of AMPK. Cell Rep 29:1511-1523.e5 (2019). PubMed: 31693892
    • Chin RM  et al. The metabolite a-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR. Nature 509:397-401 (2014). PubMed: 24828042
    • 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

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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

    The Mitotox Complex IV kit requires a blocking step. None of the other Mitotox OXPHOS kits require this block step. What characteristics of the Complex IV kit, as opposed to the I, II, and V kits, necessitates a block step?

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    Answer

    This is because the plate requires fresh blocking to prevent non specific binding of the enzyme to the plate. We believe that this occurs because CIV is a highly hydrophobic enzyme.

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