Aug 26, 2023
Mitochondria purification
- 1Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States
Protocol Citation: wusj, schekman 2023. Mitochondria purification. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldmppnl5b/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: August 03, 2023
Last Modified: August 26, 2023
Protocol Integer ID: 85959
Funders Acknowledgements:
Randy Schekman
Grant ID: Howard Hughes Medical Institute
Abstract
This protocol describes how to purify mitochondria from cell culture
Mitochondria purification
Mitochondria purification
15m
15m
HEK293T cells were trypsinized (0.05% Trypsin 00:05:00 ) and collected by centrifugation (500g 00:05:00 ).
10m
Cells were washed twice with NKM buffer (1 mM Tris HCl, pH7.3, 0.13 M NaCl, 5 mM KCl, and 7.5 mM MgCl2), and resuspended in six packed cell volumes of homogenization buffer (10 mM Tris pH 7.4, 10 mM KCl, and 0.15 mM MgCl2).
Cells were homogenized by 10 passages through a 22G needle.
Cell homogenates were mixed gently with the same volume of 2.3 M sucrose solution and centrifuged at 1200×g for 00:05:00 4 °C to remove unbroken cells and large cell debris.
5m
The recovered supernatant fractions were centrifuged at 7000×g for 00:10:00 4 °C .
10m
Mitochondria enriched in the pellet fraction were resuspended in three packed cell volumes of Mitochondria Suspension Buffer (10 mM Tris, pH 7.3, 0.15 mM MgCl2, and 0.25 mM
sucrose).