Complex I activity assay

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 6.5mg of KCN and dissolve in 1mL of 0.1Molarity (M) NaOH (stock solution of 100millimolar (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

404-877.docx

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.