Key features and details
- Assay type: Quantitative
- Detection method: Colorimetric
- Platform: Microplate reader
- Sample type: Inhibitor compounds
Product nameMitoTox™ Complete OXPHOS Activity Assay Kit (5 Assays)
Sample typeInhibitor compounds
Species reactivityReacts with: Cow, Human
MitoTox™ Complete OXPHOS Activity Assay Kit (5 assays) (ab110419) is designed for testing the direct inhibitory effect of compounds on the 5 OXPHOS complexes. Inhibitory effects of compounds on OXPHOS Complexes 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 specific Complex activity can be tested at 11 different data points in triplicate.
Complexes I, II, IV and V activity are each immunocaptured from bovine heart mitochondria by specific antibodies on 96-well microplates, and their activities measured by simple spectrophotometric assays. Complex III activity is measured by an in-solution colorimetric assay.
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.
This kit consists of the following assays:
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 5 x 96 tests Detergent 1 x 1ml 12-channel reagent reservoirs 10 units 1X Blocking Buffer 1 x 70ml 1X Complex III Mito Buffer 1 x 1ml 1X Complex IV Mito Buffer 1 x 110ml 1X Mito Buffer 2 x 5ml 1X Succinate Solution 1 x 12ml 20X Buffer 1 x 5ml 20X Wash Buffer 2 x 5ml 96-well microplate 1 unit Bovine heart mitochondria 4 x 360µl Bovine Heart Mitochondria 1 x 90µl Bovine heart mitochondria (5 mg/mL) 1 x 300µl Complex I Activity Buffer 1 x 24ml Complex II Activity Buffer 1 x 24ml Complex V Activity Buffer 1 x 24ml Cytochrome c (III) 1 x 550µl DCPIP/DCIP 1 x 250µl Detergent 1 x 20µl Detergent 2 x 100µl Blocking Powder 1 x 1.8g Phospholipids 2 x 6ml Pre-coated 96-well microplate 4 units Reagent C (Reduced Cytochrome c) 2 x 1ml Single-channel reagent reservoirs 2 units Succinate 1 x 500µl Ubiquinone 1 1 x 60µl Ubiquinone 2 1 x 60µl
Dose response curve for aurovertin, a non-competitive inhibitor of Complex V.
Typical dose response curve for oligomycin. Assay was performed following the Dose Response Assay Procedure using oligomycin, a well known Complex V inhibitor. Oligomycin was prepared in DMSO to generate a 10 mM stock. Starting with a 50 µM final concentration in well, 1:10 serial dilutions of oligomycin were generated.
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.
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.
Typical dose response curve for TTFA (2-thenoyltrifluoracetone). Assay was performed following the Dose Response Assay Procedure using TTFA, a well known Complex II inhibitor. TTFA was prepared in DMSO to generate a 100 mM stock. Starting with a 500 µM final concentration in well, 1:2 serial dilutions of TTFA were generated.
Typical dose response curve for rotenone. Assay was performed following the Dose Response Assay Procedure using rotenone, a well known Complex I inhibitor. Rotenone was prepared in DMSO to generate a 10 mM stock. Starting with a 50 µM final concentration in well, 1:10 serial dilutions of rotenone were generated.
ab110419 has been referenced in 7 publications.
- Nomiyama K et al. Targeted metabolome analysis of the dog brain exposed to PCBs suggests inhibition of oxidative phosphorylation by hydroxylated PCBs. Toxicol Appl Pharmacol N/A:114620 (2019). PubMed: 31195005
- Yi X et al. SIRT3-Dependent Mitochondrial Dynamics Remodeling Contributes to Oxidative Stress-Induced Melanocyte Degeneration in Vitiligo. Theranostics 9:1614-1633 (2019). PubMed: 31037127
- Xuan J et al. Mitochondrial dysfunction induced by leflunomide and its active metabolite. Toxicology 396-397:33-45 (2018). PubMed: 29427785
- Zhu H et al. MiR-138 protects cardiac cells against hypoxia through modulation of glucose metabolism by targetting pyruvate dehydrogenase kinase 1. Biosci Rep 37:N/A (2017). PubMed: 28899927
- Zhang C et al. Sorafenib targets the mitochondrial electron transport chain complexes and ATP synthase to activate the PINK1-Parkin pathway and modulate cellular drug response. J Biol Chem 292:15105-15120 (2017). PubMed: 28673964
- Nguyen KC et al. Mitochondrial Toxicity of Cadmium Telluride Quantum Dot Nanoparticles in Mammalian Hepatocytes. Toxicol Sci 146:31-42 (2015). PubMed: 25809595
- 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