Feb 03, 2025
Mitochondrial isolation
- 1Institut Imagine
- Team Deleidi
Protocol Citation: Maria Jose Perez J. 2025. Mitochondrial isolation. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9rjxqv3e/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: November 26, 2024
Last Modified: February 03, 2025
Protocol Integer ID: 112822
Funders Acknowledgements:
ASAP
Abstract
Mitochondrial isolation
Mitochondrial isolation
Mitochondrial isolation
Harvest and pellet 10 × 10^6 cells.
Incubate the pellet on ice for 10 minutes with 1 mL of lysis buffer supplemented with a protease inhibitor.
Centrifuge the suspension at 1000 × g for 10 minutes at 4°C.
Retain half of the supernatant as the total cellular fraction.
Subject the remaining supernatant to three additional rounds of centrifugation at 1000 × g for 10 minutes at 4°C to obtain the cleaned supernatant as the cytosolic fraction.
Resuspend the pellet from the first centrifugation step in 1.5 mL of disruption buffer supplemented with a protease inhibitor.
Mechanically disrupt the suspension by passing it through a 26-gauge needle ten times on ice.
Centrifuge the disrupted suspension at 1000 × g for 10 minutes at 4°C and discard the pellet.
Centrifuge the resulting suspension at 14000 × g for 30 minutes at 4°C.
Resuspend the final pellet enriched with mitochondria in 50 μL of Mitochondria Storage Buffer supplemented with a protease inhibitor.
Measure the protein concentration in all fractions using the Pierce‱ BCA Protein Assay Kit (Cat. number 23225, Pierce, Thermo Fisher Scientific).
Use 20 µg of protein for immunoblotting and 30 µg for measurement of protein aggregation.