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 duration
    Multiple steps standard assay
  • 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

  • Platform
    Reagents

Properties

Images

  • 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. (Note that these ranges depend on mitochondria preparation quality.) Examples of Complex I activity measured in different rat and mouse mitochondrial samples are shown in the graphs above.
  • (A) ab10972 was used to determine the Complex I activity in normal fibroblasts and rho0 fibroblasts (cells in which the mtDNA is removed and hence essential Complex I proteins are not expressed). The rho0 cells showed no/little complex I activity. In a similar analysis, (B) 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.
  • Principle of Complex I Enzyme Activity Microplate Assay Kit (ab109721)

Protocols

References

This product has been referenced in:
  • Secinaro MA  et al. Glycolysis promotes caspase-3 activation in lipid rafts in T cells. Cell Death Dis 9:62 (2018). Read more (PubMed: 29352186) »
  • Matsubara M  et al. Analysis of mitochondrial function in human induced pluripotent stem cells from patients with mitochondrial diabetes due to the A3243G mutation. Sci Rep 8:949 (2018). Read more (PubMed: 29343702) »

See all 57 Publications for this product

Customer reviews and Q&As

Filter by Ratings

Complex I activity in HCT116 cells

Excellent Good 4/5 (Ease of Use)
Abreviews
HCT116 cells were cultivated in standard DMEM medium for 48 hrs. Cell preparation was performed as described in the protocol. Protein concentration in each sample was quantified and adjusted by Bradford assay.
We looked at complex I activity dependent on the protein amount applied to the plate (fig.) thus we would suggest to use protein amounts around 200 µg for this assay.
The kit is easy to use but takes a long time to perform. The results seem to be reliable.
Username

Mr. Christian Marx

Verified customer

Submitted Dec 12 2013

Abreviews
Very easy to follow instructions and to standardize using cell lysates. Good specificity with microplane reader.
Username

Abcam user community

Verified customer

Submitted Feb 28 2018

Abreviews
I used isolated protein from guinea pig placenta to perform the experiment with this kit. Complex I enzyme activity can be detected.
Username

Abcam user community

Verified customer

Submitted Feb 07 2017

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.
Username

Dr. Amit Agarwal

Verified customer

Submitted Jul 24 2013

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

Sign up