| Assay Method Information | |
| | Cytochrome b Enzyme Assay |
| Description: | The cytochrome b enzyme assay was adapted from previous studies (Gutierrez-Cirlos et al., 2004). Briefly, compounds were diluted in a 23 reaction buffer containing 100 mM Tris-Cl (pH 7.4), 10 mM sodium azide, 0.02% BSA, and 0.1% Tween 20. Mitochondria (~5 mg protein) and cytochrome c (final concentration50 mM) were then added, and the reaction was initiated by the addition of ubiquinol (50 mM), reduced from ubiquinone with dithionite. Reaction plates were incubated at room temperature for 10 min before reading absorbance at 550 nm. Absolute IC50 values were calculated in GraphPad Prism 6.0. Antimycin A (200 nM) was included on every plate as a control for 100% inhibition. |
| Affinity data for this assay | |
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