Mitochondria complex 3 activity assay kit

Intact mitochondria were isolated from HeLa cells by using the Qproteome Mitochondria isolation kit (Qiagen, Cat. No 37612) according to the manufacturer’s manual. The complex III activity was measured by using the Mitochondria Complex III Activity Assay Kit (Biovision, Cat. No. K520). In brief, we added cytochrome c in samples containing 3–6 µg of isolated mitochondria and recorded the absorbance of the reduced cytochrome c at 550 nm, at 30-s intervals for 10 min at RT. Antimycin A inhibitor and DMSO were used in negative and background control samples, respectively. Complex III specific activity was calculated by applying the following formula: $$\text{Net}\text{complex}\text{III}\text{specific}\text{activity}=\text{Complex}\text{III}\text{specific}\text{activity}\text{w}/\text{o}\text{Antimycin}\text{A}-\text{Complex}\text{III}\text{specific}\text{activity}\text{with}\text{Antimycin}\text{A}.$$
Complex III specific activity was calculated by applying the following equation: $$\text{Complex}\text{III}\text{specific}\text{activity}=\mathrm{\Delta }\text{C}/\left(\mathrm{\Delta }\text{t}\ast \text{p}\right)\ast \text{D}$$ where ∆C = change in reduced cytochrome c concentration during the ∆t; ∆t = t2 − t1 (min); p = mitochondria protein sample (µg); D = dilution factor.

Mitochondria were isolated from the DGUOK deficient rat tissues using mitochondria isolation kit for tissue (Abcam, #ab110168). The activities of electron transport chain complex I, II, III, IV, V were determined using complex I enzyme activity microplate assay kit (Abcam, #ab109721), complex II enzyme activity microplate assay kit (Abcam, #ab109908), mitochondria complex III activity assay kit (Biovision, #K520), complex IV rodent enzyme activity microplate assay kit (Abcam, #ab109911), and ATP synthase enzyme activity microplate assay kit (Abcam, #ab109714), respectively, following manufacturer’s instruction.