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The MitoTox™ OXPHOS Complex III Activity Kit is designed for screening the direct inhibitory effects of compounds on Complex II and III activity. Bovine heart mitochondria are provided in this kit as they are a rich source of these two enzymes. The kit contains succinate (Solution 1) which is the electron donor of Complex II, as well as cytochrome c (oxidized), the electron acceptor of Complex III. Ubiquinone, the electron acceptor of Complex II, is present in the mitochondria. The rate of the coupled Complex II+III reaction is measured by monitoring the conversion of cytochrome c in its oxidized form, cytochrome c (oxidized), to its reduced form, cytochrome c (reduced), as a linear increase in absorbance at exactly 550 nm (reading the absorbance at 540 nm will not be sufficient for this assay). The kit requires the use of rotenone and KCN (not supplied with the kit). Addition of rotenone, an inhibitor of Complex I, ensures that all reduction of Cytochrome c is via Complex II. Addition of KCN ensures that there is no re-oxidation of Cytochrome c by Complex IV. The user may add antimycin, an inhibitor of Complex III activity, in the screening procedure as a positive control. 50% inhibition of Complex III activity is obtained with 22 ± 4 nM antimycin at assay conditions described in the protocol.
Store at -80°C: Bovine Heart Mitochondria, Cytochrome c (oxidized).
Store at 4°C: Succinate Solution, Microplate, Mito Buffer and Reagent Reservoirs: Reagent Reservoirs may be stored at room temperature.
|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 (oxidized)||1 x 550µl|
|Single-channel reagent reservoirs||2 units|
Our Abpromise guarantee covers the use of ab109905 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Functional Studies||Use at an assay dependent dilution.|