MitoTox™ Complex II + III OXPHOS Activity Assay Kit

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. **Related products** Review the , or the full 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.