Mar 30, 2023

Public workspaceComplex I activity assay

DOI
dx.doi.org/10.17504/protocols.io.4r3l27r8qg1y/v1
  • 1German Center for Neurodegenerative Diseases (DZNE), Tübingen, 72076 Germany
Federico Bertoli
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DOI: dx.doi.org/10.17504/protocols.io.4r3l27r8qg1y/v1
Protocol Citation: María José Pérez J., michela.deleidi 2023. Complex I activity assay. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l27r8qg1y/v1
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: March 28, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 79543
Keywords: Complex I activity assay, - Extinction coefficient, Enzyme activity, ASAPCRN
Abstract
This protocol describes the complex I activity assay.
Attachments
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404-877.docx
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Guidelines
Reference for analysis: https://www.nature.com/articles/nprot.2012.058
Materials
KIT:
Mitocheck Complex I activity assay kit ReagentMitoCheck® Complex I Activity Assay KitCayman Chemical CompanyCatalog #700930
ReagentQproteome Mitochondria Isolation KitQiagenCatalog #37612

Before start
  • All assays are carried out at Temperature25 °C .
  • After mitochondrial isolation (Qproteome Mitochondria Isolation Kit. QIAGEN Cat. No. / ID: 37612), resuspend the final pellet in Amount50 µL of storage buffer, keep isolated mitochondria TemperatureOn ice .
  • In a ventilated hood, weigh out Amount6.5 mg of KCN and dissolve in Amount1 mL of Concentration0.1 Molarity (M) NaOH (stock solution of Concentration100 millimolar (mM) )
  • Label two polystyrene tubes as A and B. For 20 reactions prepare:
AB
Tube A (1 ml)Tube B (675 µl)
910 µl of Complex I activity buffer625 µl of Complex I activity buffer
20 µl of 100mM KCN (1 mM)30 µl of NADH assay reagent
50 µl FF-BSA Assay Reagent20 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 Amount50 µL of the contents of tube A to each well.

Pipetting
Add Amount20 µL of sample to each well.

Pipetting
Place plate in plate reader and add Amount30 µL of B to each well.
Pipetting
Immediately measure absorbance at 340 nm in kinetic read mode (30 seconds intervals for Duration00:05:00 at Temperature25 °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)].
Extinction coefficient for NADH 6.2 mM-1 cm-1.