Sep 23, 2023
Mitochondrial isolation protocol
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna (AT)
Protocol Citation: Elias Adriaenssens 2023. Mitochondrial isolation protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3x4zzg25/v1
Manuscript citation:
The methodology is based on Wieckowski et al. 2009 Nat Protocols.
Wieckowski, M., Giorgi, C., Lebiedzinska, M.et al. Isolation of mitochondria associated membranes and mitochondria from animal tissues and cells. Nat Protoc 4, 1582–1590 (2009).
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: June 26, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 84020
Keywords: Mitochondrial isolation, HeLa cells, ASAPCRN, crude mitochondrial fractions from hela cell, mitochondrial isolation protocol, mitochondrial isolation protocol this protocol, crude mitochondrial fraction, hela cell
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
Marie Skłodowska-Curie MSCA Postdoctoral fellowship
Grant ID: 101062916
Abstract
This protocol describes how to isolate crude mitochondrial fractions from HeLa cells.
Attachments
752-1920.pdf
50KB
Materials
MATERIALS
Mitochondrial isolation buffer:
| A | B | |
| HEPES (pH 7.4) | 5 mM | |
| mannitol | 250 mM | |
| EGTA | 0.5 mM |
make fresh on the day itself!
Oligomycin/Antimycin A cocktail:
| A | B | |
| Oligomycin | 10 µM | |
| Antimycin A | 4 µM |
In case cells were treated for more than 8 hours, we also added 10 µM Q-VD-OPh (A1901, ApexBio) to suppress apoptosis.
RIPA buffer:
| A | B | |
| Tris-HCl pH 8.0 | 50 mM | |
| NaCl | 150 mM | |
| sodium deoxycholate | 0.50% | |
| SDS | 0.10% | |
| NP-40 | 1% |
supplemented by cOmplete EDTA-free protease inhibitors (11836170001, Roche) and phosphatase inhibitors (Phospho-STOP, 4906837001, Roche).
Oligomycin AApexBio TechnologyCatalog #A5588
Antimycin A from Streptomyces sp.Merck MilliporeSigma (Sigma-Aldrich)Catalog #A8674
Q-VD-OPhApexBio TechnologyCatalog #A1901
cOmplete™, Mini, EDTA-free (Protease Inhibitor)RocheCatalog ##11836170001)
Roche PhosSTOP™Merck MilliporeSigma (Sigma-Aldrich)Catalog #4906837001
Mitochondrial isolation
1h 15m
Seed HeLa cells in 15 cm dishes and grow until confluence. Treat the cells with DMSO or the mitophagy-inducing cocktail Oligomycin/Antimycin A (O/A) if needed.
Collect HeLa cells by trypsinization and resuspension in DMEM medium. Centrifuge the cells at 300 x g, 4°C, 00:10:00 .
10m
Resuspend the cell pellet in 1 mL of ice-cold PBS (1x) and spin again for 300 x g, 4°C, 00:05:00 .
5m
Remove the PBS and resuspend the cell pellet in 1 mL mitochondrial isolation buffer, which was cooled to 4 °C .
Note
Note that the mitochondrial isolation buffer is prepared fresh on the day itself.
To lyse cells without damaging the mitochondria, pipet the 1 mL cell suspension up and down (15-20x) with a 26.5G needle.
Note
This should lyse the plasma membrane but leave organelles intact.
Spin the suspension down at 600 x g, 4°C, 00:10:00 .
10m
Keep the supernatant and centrifugation 600 x g, 4°C, 00:10:00 .
Note
The mitochondria are located in the supernatant at this speed, the pellet contains intact cells, cell nuclei, and other large cell debris.
10m
After two spins at 600 x g , subject the supernatant to 7000 x g, 4°C, 00:10:00 .
10m
The pellet contains the mitochondria, so remove the supernatant and resuspend the pellet in 1 mL mitochondrial isolation buffer.
Then, centrifugation 7000 x g, 4°C, 00:10:00 .
Note
The supernatant from the first spin at 7000 x g can be stored as a cytosolic fraction.
10m
After two spins at 7000 x g , subject the resuspended pellet to 10000 x g, 4°C, 00:10:00 .
10m
The pellet contains the mitochondria, so remove the supernatant and resuspend the pellet in 1 mL mitochondrial isolation buffer.
Then, centrifugation 10000 x g, 4°C, 00:10:00 .
10m
After two spins at 10000 x g , the pellet can be resuspended in RIPA lysis buffer or used for further procedures such as mitochondrial import assays, proteinase K protection assays, etc.