Key features and details
- Assay type: Direct
- Detection method: Colorimetric
- Platform: Microplate reader
- Assay time: 5 hr
Product nameMitoTox™ Complex IV OXPHOS Activity Assay Kit
See all Complex IV kits
Assay time5h 00m
Species reactivityReacts with: Cow, Human
MitoTox™ Complex IV OXPHOS Activity Assay Kit (ab109906) is designed for testing the direct inhibitory effect of compounds on Complex IV activity in only 5 hours. Complex IV extracted from the provided bovine heart mitochondria (a rich source of Complex IV) is immunocaptured by specific antibodies on the plate. Complex IV activity can be observed as decrease in absorbance at OD 550 nm. The intra-assay and inter-assay variation of this assay are both <10%.
Inhibitory effects of compounds on Complex IV 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 IV 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 Reagent C 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 Blocking Buffer 1 x 70ml 1X Complex IV Mito Buffer 1 x 110ml Bovine Heart Mitochondria 1 x 90µl Detergent 1 x 20µl Blocking Powder 1 x 1.8g Pre-coated 96-well microplate 1 unit Reagent C (Reduced Cytochrome c) 2 x 1ml
- Cytochrome c oxidase
- Cytochrome oxidase
Typical dose response curve for KCN. Assay was performed following the Dose Response Assay Procedure using KCN, a well known Complex IV inhibitor. KCN was prepared in 0.1 M NaOH to generate a 100 mM stock. Starting with a 100 µM final concentration in well, 1:10 serial dilutions of KCN were generated.
ab109906 has been referenced in 4 publications.
- Baccelli I et al. Mubritinib Targets the Electron Transport Chain Complex I and Reveals the Landscape of OXPHOS Dependency in Acute Myeloid Leukemia. Cancer Cell 36:84-99.e8 (2019). PubMed: 31287994
- Wang Y et al. Metformin Improves Mitochondrial Respiratory Activity through Activation of AMPK. Cell Rep 29:1511-1523.e5 (2019). PubMed: 31693892
- Chin RM et al. The metabolite a-ketoglutarate extends lifespan by inhibiting ATP synthase and TOR. Nature 509:397-401 (2014). PubMed: 24828042
- Lai K et al. Integrated Compound Profiling Screens Identify the Mitochondrial Electron Transport Chain as the Molecular Target of the Natural Products Manassantin, Sesquicillin, and Arctigenin. ACS Chem Biol N/A:N/A (2012). PubMed: 23138533