License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 27, 2022
Last Modified: March 17, 2023
Protocol Integer ID: 61490
Keywords: Complex I activity assay, - Extinction coefficient, Enzyme activity
Abstract
This protocol describes the complex I activity assay.
After mitochondrial isolation (Qproteome Mitochondria Isolation Kit. QIAGEN Cat. No. / ID: 37612), resuspend the final pellet in 50 µL of storage buffer, keep isolated mitochondria On ice.
In a ventilated hood, weigh out 6.5 mg of KCN and dissolve in 1 mL of 0.1 Molarity (M) NaOH (stock solution of 100 millimolar (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
Protocol
Protocol
5m
5m
Distribute the contents of tube A and B in strips suitable for multichannel use.
In a Half Volume 96-well clear plate add 50 µL of the contents of tube A to each well.
Add 20 µL of sample to each well.
Place plate in plate reader and add 30 µL of B to each well.
Immediately measure absorbance at 340 nm in kinetic read mode (30 seconds intervals for 00:05:00 at 25 °C)
5m
Calculations
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)].