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 72 Publications for this product

Customer reviews and Q&As

11-20 of 86 Abreviews or Q&A

30ug Lung mitochondrion samples in buffer containing 250 mM sucrose, 20 mM HEPES, 1 mM EDTA, 1 mM EGTA, 0.25% protease inhibitor, and 0.5% BSA, pH 7.4 were used for the assay.

Dr. Amit Agarwal

Verified customer

Submitted Jul 24 2013

Question
Answer

Red blood cells do not contain mitochondria so isolation of peripheral blood mononuclear cells (PBMC) is recommended. Once the required amount of cells (number or mass) is isolated I would not anticipate any specific problems.

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Answer

The protocol suggest a sample loading of 100ug/well (with is equivalent to 0.5mg/mL on a 200uL volume loading). According to what the customer says they only have 8uL at a concentration of 5.5mg/mL. After adding the detergent and centrifuging, they would need to dilute the sample to 500ug/mL (1/10 dilution), which will only give them 80uL for loading and not 200uL. The customer really needs more material to be able to test the sample at least in duplicates and ideally at a couple of concentrations.

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Question
Answer

You can use total tissue homogenate or cultured cell lysate. These will have lower activity per mass of protein of course.

Figures 2 and 3 show activity derived from whole cell lysates in cultured HepG2 (human liver derived), human fibroblasts and rat cardiomyocytes.

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Answer

In step 6.4, the mitochondria will be lysed. Step 6.5 is required to pellet insolubles post-lysis. Save the pellet but it should not contain anything of value assuming lysis was successful.

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Answer

Thank you for your enquiry and interest in our products.

I can confirm that enzymatic digestion of the cultured cells with trypsin+EDTA does not interfere with this particular assay (ab109721: Complex I Enzyme Activity Microplate Assay Kit) as long as the trypsin is quenched soon after the cells detached from the plate and a good viability count is obtained.

If you need any further assistance in the future, please do not hesitate to contact me.

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Answer

Thank you for contacting us.
We following kits available for complex III. DO you think this the one you are looking for;
https://www.abcam.com/mitotox™-complex-iii-oxphos-activity-microplate-assay-ab109905.html
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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Question
Answer


I've confirmed with the kit development scientists that you can prepare samples for both kits at the same time.

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Answer

Thank you for contacting us.

If the stating protein concentration is low, the detergent can be scaled down to use less, so instead of 10X dilution, 20X or 40X dilution could be used but I wouldn’t go below 40X.

If the sample is already extracted in detergent there isn’t much to do but perhaps add more to the well, 250-200uL and incubate longer.

Please let me know if you have any further questions!

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Answer


We haven't specifically tested the complex I or II kits under reducing conditions and cannot guarantee that it will not affect the enzyme activities. With that being said, my colleagues believe that a reducing agent at moderate concentration shouldn't affect the assay capture step significantly, especially if the reduced samples are sufficiently diluted in the wells.

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11-20 of 86 Abreviews or Q&A

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