Key features and details
- Assay type: Direct
- Detection method: Colorimetric
- Platform: Microplate reader
- Assay time: 30 min
Product nameMitoTox™ Complex II + III OXPHOS Activity Assay Kit
See all Complex III kits
Assay time0h 30m
Species reactivityReacts with: Cow, Mammals
MitoTox™ Complex II+ III OXPHOS Activity Assay Kit (ab109905) is designed for testing the direct inhibitory effect of compounds on Complex III activity in only 30 minutes. The assay is performed using whole bovine heart mitochondria, a rich source of OXPHOS complex. Succinate (electron donor of Complex II) and oxidized cytochrome c (electron acceptor of Complex III) are added to the mitochondria to start the electron transfer pathway that takes place during oxidative phosphorylation. The rate of couple Complex II + III reaction is measured by monitoring the conversion of oxidized cytochrome c into reduced form, which can be observed as increase in absorbance at OD 550 nm. The assay requires rotenone and KCN (not supplied): rotenone (Complex I inhibitor) blocks electron transfer to ubiquinone, ensuring that all reduction of cytochrome c happens via Complex II. The addition of KCN (Complex IV inhibitor) ensures that there is no re-oxidation of cytochrome c. The intra-assay and inter-assay variation of this assay are both < 10%.
Inhibitory effects of compounds on Complex III 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 III 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.
Store Bovine Heart Mitochondria and Cytochrome c (oxidized) at -80°C. Store all other components at 4°C.
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.
Storage instructionsPlease refer to protocols.
Components 96 tests 12-channel reagent reservoirs 2 units 1X Complex III Mito Buffer 1 x 1ml 1X Succinate Solution 1 x 12ml 96-well microplate 1 unit Bovine heart mitochondria (5 mg/mL) 1 x 300µl Cytochrome c (III) 1 x 550µl Single-channel reagent reservoirs 2 units
RelevanceComplex II (succinate-ubiquinone oxidoreductase), one of the mitochondrial respiratory chain complexes, transfers electrons from succinate generated during the citric acid cycle to Complex III (ubiquinolcytochrome c oxidoreductase), via a mobile electron shuttle, ubiquinone. Complex III transfers electrons to Complex IV (cytochrome c oxidase) via another mobile electron shuttle, cytochrome c.
- coenzyme Q : cytochrome c — oxidoreductase
- cytochrome bc1 complex
Typical dose response curve for antimycin A. Assay was performed following the Dose Response Assay Procedure using antimycin A, a well known Complex III inhibitor. Antimycin A was prepared in DMSO to generate a 10 mM stock. Starting with a 150 µM final concentration in well, 1:4 serial dilutions of antimycin A were generated.
Mitchondiral fractions from the heart tissue of Rats infected by S. pneumoniae, or given PBS sham control, were subjected to measurements of complex I-V activities. Complex I was measured with ab109721 (top left), Complex II + III were measured using ab109905 (top right), Complex IV was measured using ab109911 (bottom left) and Complex V was measured using ab109714 (bottom right).
Freshly isolated mitochondrial pellets were resuspened in PBS supplemented with 10% detergent provided in the kits. Protein concentrations of these mitochondrial lysates were estimated and 25 μg (for complex I, IV and V) or 100 μg (for complex II+III) mitochondrial protein was used per reaction. Enzyme activities were measured spectrophotometricly in triplicate and expressed as changes of absorbance per minute per mg protein
ab109905 has been referenced in 23 publications.
- Lee DH et al. Withania somnifera Extract Enhances Energy Expenditure via Improving Mitochondrial Function in Adipose Tissue and Skeletal Muscle. Nutrients 12:N/A (2020). PubMed: 32046183
- Subedi A et al. A novel inhibitor of tumorspheres reveals the activation of the serine biosynthetic pathway upon mitochondrial inhibition. FEBS Lett 593:763-776 (2019). PubMed: 30874300
- Agarwal E et al. Myc-mediated transcriptional regulation of the mitochondrial chaperone TRAP1 controls primary and metastatic tumor growth. J Biol Chem 294:10407-10414 (2019). PubMed: 31097545
- Caldwell CC et al. Treadmill exercise rescues mitochondrial function and motor behavior in the CAG140 knock-in mouse model of Huntington's disease. Chem Biol Interact 315:108907 (2019). PubMed: 31778667
- Wang Y et al. Metformin Improves Mitochondrial Respiratory Activity through Activation of AMPK. Cell Rep 29:1511-1523.e5 (2019). PubMed: 31693892