Complex I Enzyme Activity Microplate Assay Kit (Colorimetric) (ab109721)

Overview

  • Product name
    Complex I Enzyme Activity Microplate Assay Kit (Colorimetric)
    See all Complex I kits
  • Detection method
    Colorimetric
  • Sample type
    Cell culture extracts, Tissue
  • Assay type
    Enzyme activity
  • Assay time
    3h 30m
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human
  • Product overview

    Complex I Enzyme Activity Assay Kit (ab109721) is a kit designed for the analysis of mitochondrial OXPHOS Complex I enzyme activity from human, rat, mouse and bovine cell and tissue extracts.
    Capture antibodies specific for Complex I are pre-coated in the microplate wells. Samples are added to the microplate wells which have been precoated with a specific capture antibody. After the target has been immobilized in the well, Complex I activity is determined by following the oxidation of NADH to NAD+ and the simultaneous reduction of a dye which leads to increased absorbance at OD=450 nm. By analyzing the enzyme's activity in an isolated context, outside of the cell and free from any other variables, an accurate measurement of the enzyme's functional state can be understood.


    Complex I activity is controlled by enzyme amount and by post-translational phosphorylation at key specific regulatory residues. Cellular metabolism governs these two factors. Ultimately, the cell type and growth conditions will affect Complex I activity measurements.


    Note: This activity assay measures the diaphorase-type activity of Complex I. This activity is not dependant on the presence of ubiquinone and therefore inhibitors, such as rotenone, which bind at or near the ubiquinone binding site do not inhibit this assay. However, both the activity assay and the quantity assay described here are affected by enzyme assembly deficiencies.


    Review our Metabolism Assay Guide to learn about assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress, and also about how to assay metabolic function in live cells using your plate reader.

  • Notes

    ab109721 is shipped at 4°C. 20X NADH and 100X Dye are shipped lyophilized. Rehydrate 20X NADH by adding 1.1 mL H2O. Rehydrate 100X Dye by adding 0.25 mL H2O, then vortex each thoroughly until dissolved. After hydration unused amounts of these two materials should be stored at -80°C. When planning multiple experiments, aliquot these reagents to prevent freeze thaw cycles. 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

  • ab109721 measures Complex I activity in human cultured cells within the recommended ranges given in the protocol. Example of Complex I activity measured in Hep2 cells is showed. (Note that these ranges depend on mitochondria preparation quality).

  • ab109721 measures Complex I activity in human and rat cultured cells but also in tissues/tissue mitochondria samples within the recommended ranges given in the protocol. Examples of Complex I activity measured in different rat tissue mitochondrial samples are shown. (Note that these ranges depend on mitochondria preparation quality).

  • Examples of Complex I activity measured in different rat tissue mitochondrial samples are shown. (Note that these ranges depend on mitochondria preparation quality).

  • (A) ab109721  was used to measure Complex I activity in normal and Rho0 human fibroblast whole cell lysates at 1mg/mL. Rho0 cells are cells in which the mitochondrial DNA has been removed and therefore essential Complex I proteins are not expressed. As shown, the rho0 cells showed no/little complex I activity.

    (B) In a similar analysis, rat cardiomyocytes were grown for 5 days in ± 40 µM chloramphenicol (CAM) to inhibit mitochondrial protein synthesis, Complex I assembly and hence activity was greatly reduced in samples loaded at 0.5 mg/mL whole cell lysates.

  • Principle of Complex I Enzyme Activity Microplate Assay Kit (ab109721)

Protocols

References

This product has been referenced in:
  • Zhang XL  et al. RNAi-mediated knockdown of DJ-1 leads to mitochondrial dysfunction via Akt/GSK-3ß and JNK signaling pathways in dopaminergic neuron-like cells. Brain Res Bull N/A:N/A (2019). Read more (PubMed: 30634017) »
  • Xu D  et al. Novel role of mitochondrial GTPases 1 in pathological cardiac hypertrophy. J Mol Cell Cardiol 128:105-116 (2019). Read more (PubMed: 30707992) »
See all 74 Publications for this product

Customer reviews and Q&As

81-86 of 86 Abreviews or Q&A

Answer

Thank you for contacting us with your questions and for your patience. I forwarded your questions to the lab at Mitosciences, and I received the following information. 1) This kit has not been tested with drosophila samples, so species cross reactivity is unknown. The location of the epitope is also not known, so it is difficult to predict cross reactivity even though the proteins are 80% similar. 2) The advantage of this kit over in-solution CI activity assay is that by immunocapturing the enzyme, you remove other enzymes in the sample extract with dehydrogenase (diaphorase) activity present in the cytoplasm, which create a high background in the in-solution assay. Typically this is why it became standard in the mitochondria field to always perform an in-solution activity with the Complex I specific inhibitor rotenone even when testing mitochondrial fractions- to know what percentage of NADH dehydrogenase activity in a sample actually came from Complex I. The immunocapture step simply removes the need to isolate mitochondria (which is very tedious) and the need to test with a highly toxic inhibitor. Rotenone inhibits specifically the ubiquinone reaction of complex I. Ab109721 only tests half of the reaction of Complex I (NADH Dehydrogenase). For inhibition assays, the kit ab109903 can be used, which uses a different capture antibody against CI and it comes with the sample (bovine heart mitochondria), so the user can test whether a compound inhibits or not CI in vitro. The mechanics of the assay are different and are geared towards toxicity testing. CI is not only affected by pharmacological inhibition, but also by subunit composition, issues with assembly of the holocomplex and even post-translational modifications. This type of activity has been used for some time in diagnostics and in particular when mitochondrial isolation is not possible due to low/impure yield.  Researchers purchase this kit because it is high throughput, simpler, and faster than the conventional in-solution assay after mitochondrial extraction.    I hope this information is helpful, but please let me know if you have any further questions and we will be happy to help.

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Answer

Thank you for contacting us with your questions. I forwarded your questions to the lab at Mitosciences to ask for their input, and this is their reply: "Unfortunately there are no specific inhibitors of this part of the complex I reaction.  Evidence we have for specificity: (1) Antibody used has been exhaustively validated for Complex I enzyme IP purity by mass spec (2) Unrelated antibodies give absolutely no signal (3) Cells treated with a variety of agents to remove mtDNA or mtDNA encoded proteins results in no activity (e.g. rho0, NRTI (ddC) treated, or antibiotic (chlorampheniocol) treated cells (4) Cell lines analyzed by this antibody/reaction scheme were successfully identified as patient lines with enzymatic deficiencies" I hope this information is helpful, but please let me know if you have any further questions.

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Answer

Thank you for contacting us. The lab confirms that samples can be frozen/thawed. I have asked about alternatives to chloramphenicol treatment for negative controls. Two that I found from an online search for diaphorase inhibition were bathocuproine and dicoumarol/dicumarol, a copper-specific chelating agent and an anticoagulant, respectively. The article at the following link discusses a few others: Molecular characterization of binding of substrates and inhibitors to DT-diaphorase: combined approach involving site-directed mutagenesis, inhibitor-binding analysis, and computer modeling. Mol Pharmacol. 1999 Aug;56(2):272-8. http://molpharm.aspetjournals.org/content/56/2/272.full I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for your inquiry. The answers to your questions are below: 1) Yes an overnight incubation at 4C will work but protease inhibitors are recommended for any overnight incubation. 2) When we rinse, there is no need to wait, but it won't affect the assay to pause for a few moments. You can read the plate right away. 3) Isolated frozen mitochondria will work well and fresh is not necessary. I hope this information helps. Please contact us with any other questions.

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Answer

Thank you for your recent telephone enquiry. I am sorry it has taken a little time to confirm this information for you. I can confirm that in this procedure, samples are all brought to the same concentration in Step 3, before they are lysed in Step 4, as described in the protocol. After extraction, the samples can be protein assayed again and some people like to do this but it should not be necessary given that the samples are all at the same concentration and prepared the same as instructed in Step3). I hope this will be helpful to you. Should you have any futher questions, please do not hesitate to contact us.

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Question
Answer

Thanks for your call yesterday and for your patience while I have been in touch with my colleagues at MitoSciences. To prepare cell extracts to use in the kit, they collect the cells in PBS and dilute to 5.5 mg/mL before the detergent is added. You can follow the "Preparation of Samples" section of the protocol exactly as stated, with the addition of collecting the cells in PBS. I hope this information will be helpful, but please let me know if you have any further questions.

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81-86 of 86 Abreviews or Q&A

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