Complex I activity assay
michela.deleidi, Maria Jose Perez J.
Abstract
This protocol describes the complex I activity assay.
Before start
All assays are carried out at 25°C.* After mitochondrial isolation (Qproteome Mitochondria Isolation Kit. QIAGEN Cat. No. / ID: 37612), resuspend the final pellet in 50µL of storage buffer, keep isolated mitochondria On ice.
- In a ventilated hood, weigh out
6.5mgof KCN and dissolve in1mLof0.1Molarity (M)NaOH (stock solution of100millimolar (mM)) - Label two polystyrene tubes as A and B. For 20 reactions prepare:
| A | B |
|---|---|
| Tube A (1 ml) | Tube B (675 µl) |
| 910 µl of Complex I activity buffer | 625 µl of Complex I activity buffer |
| 20 µl of 100mM KCN (1 mM) | 30 µl of NADH assay reagent |
| 50 µl FF-BSA Assay Reagent | 20 µl of Ubiquinone assay reagent |
| 20 µl of Vehicle |
Attachments
Steps
Protocol
Distribute the contents of tube A and B in strips suitable for multichannel use.
In a Half Volume 96-well clear plate add 50µL of the contents of tube A to each well.
Add 20µL of sample to each well.
Place plate in plate reader and add 30µL of B to each well.
Immediately measure absorbance at 340 nm in kinetic read mode (30 seconds intervals for 0h 5m 0s at 25°C)
Calculations
The specific activity of complex I is calculated as nmol min−1 mg−1 of protein according to the following equation:
Enzyme activity (nmol min−1 mg−1) = (Δ Absorbance/min × 1,000)/[(extinction coefficient × volume of sample used in ml) × (sample protein concentration in mg ml−1)].
Extinction coefficient for NADH 6.2 mM-1 cm-1.
