Hello. We're improving abcam.com and we'd welcome your feedback.

Hello. We're improving abcam.com and we'd welcome your feedback.

Infomation icon

We haven't added this to the BETA yet

New BETA website

New BETA website

Hello. We're improving abcam.com and we'd welcome your feedback.

Take a look at our BETA site and see what we’ve done so far.

Switch on our new BETA site

Now available

Search and browse selected products

  • A selection of primary antibodies

Purchase these through your usual distributor

In the coming months

  • Additional product types
  • Supporting content
  • Sign in to your account
  • Purchase online

MitoTox™ Complex IV OXPHOS Activity Assay Kit (ab109906)

Submit a review Q&A (2)References (5)
MitoTox™ Complex IV OXPHOS Activity Assay (ab109906)

    Key features and details

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

    Overview

    • Product name

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

    • Detection method

      Colorimetric

    • Assay type

      Direct

    • Assay time

      5h 00m

    • Species reactivity

      Reacts with: Cow, Human

    • Product overview

      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. 


       


       

    • Notes

      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.

    • Platform

      Microplate reader

    Properties

    Images

    • 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.

    References (5)

    Publishing research using ab109906? Please let us know so that we can cite the reference in this datasheet.

    ab109906 has been referenced in 5 publications.

    • Papachristodoulou A  et al. NKX3.1 Localization to Mitochondria Suppresses Prostate Cancer Initiation. Cancer Discov 11:2316-2333 (2021). PubMed: 33893149
    • 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

    Customer reviews and Q&As

    Show All Reviews Q&A
    Submit a review Submit a question

    1-2 of 2 Abreviews or Q&A

    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?

    Read More
    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).

    Read More

    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?

    Read More
    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.

    Read More

    Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
    For licensing inquiries, please contact partnerships@abcam.com