Mar 13, 2025
Mitochondrial Sub-fractionation Using Differential Centrifugation - Cells
- 1Hertie Institute for Clinical Brain Research
Protocol Citation: Julia Fitzgerald 2025. Mitochondrial Sub-fractionation Using Differential Centrifugation - Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzkwpxvx1/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 13, 2025
Last Modified: March 13, 2025
Protocol Integer ID: 124326
Keywords: mitochondrial fractionation, crude mitochondria, enrichment, isolated
Disclaimer
NA
Abstract
Crude fractionation of cells to yield crude mitochondria and subfractions using differential centrifugation
Guidelines
GM cells should be treated according to local safety guidelines
Materials
Eppendorf tubes
Dounce tissue grinder (loose and tight fitting)
Tris
EDTA
Sucrose
Centrifuge capable of 10,000g
Ultracentrifuge (optional)
Protocol materials
Safety warnings
No warnings
Ethics statement
NA
Before start
Prepare and cool the buffers
Prepare ice
Process collected cells and prepare crude mitochondria
Process collected cells and prepare crude mitochondria
2h 5m
2h 5m
Start with a cell pellet (for crude mitochondrial preparation start with a minimum of a confluent 10cm dish, for sub-fractionation start with minimum of 3-6 confluent T75cm flasks). The cell pellet is always kept cold On ice
Wash the cell pellet once with 1X PBS (Phosphate-buffered saline ) and 1200 rpm, 21°C, 00:05:00 to generate a washed cell pellet. On ice
5m
Dislodge the cell pellet with approximately 1000 µL of TES buffer (70 mM Tris base, 1mM EDTA, 0.25 M sucrose, pH 7.4) and transfer to a loose fit hand held homogenizer (such as Eppi-pestles +1.5mL tube). With strong force by hand, turn the pestles ten times. Transfer the homogenate to a tight fitting glass homogenizer (such as dounce tissue grinder Sigma-Aldrich P1110-1EA) and with strong force by hand dounce the homogenate by turning the glass pestle as you move up and down in the glass mortar tube 10 times. On ice
Equipment
Dounce Tissue Grinder Set with Two Glass Pestles
NAME
Wheaton
BRAND
cat# 357538
SKU
LINK
Equipment
Eppi-pestles
NAME
Pestle for 1.5mL tube
TYPE
Schuett biotec
BRAND
3200512
SKU
LINK
Transfer the homogenate to a 2mL round bottomed "Eppendorf" tube (Eppi). Optional: aliquot a small amount of the homogenate in a new tube labelled "TE" total extract or homogenate. On ice
1000 x g, 4°C, 00:10:00
10m
Carefully remove the supernatant and transfer to a fresh 1.5mL Eppi and label "M" On ice . Add 500µL TES buffer to the "nuc" pellet and homogenize again (see above). 1000 x g, 4°C, 00:10:00 . Transfer the supernatant to the "M" tube on ice. Label the nuc pellet tube "N" and store if needed. On ice
10m
Tube "M" > 9800 x g, 4°C, 00:15:00
15m
Transfer the supernatant to a new Eppi labelled "C" for cytosolic fraction. On ice
Look carefully at the "M" pellet which is crude mitochondria. The pellet should be red/brown/yellow in colour and flaky/hydrophobic (see image in tab "description" of this protocol). If the mitochondrial pellet is surrounded by contamination (white fluffy pellet), the contaminating white fluffy pellet can be carefully removed with a pasteur pippette pump or a pipette.
Optional: wash the mitochondrial pellet in TES buffer and centrifuge again 9800 x g, 4°C, 00:10:00
10m
Resuspend the mitochondrial pellet in TMG buffer (10 mM Tris, 5 mM MgCl2, 20 % (v/v) glycerol) and perform protein estimation. Working range: minimum 0.7mg/mL mitochondrial protein concentration for most applications and assays. On ice
Optional: aliquot a small amount of the crude mitochondrial extract. On ice
Optional: to store the crude mitochondrial fraction for further use; 9800 x g, 4°C, 00:15:00 and store as a dry pellet at -80 °C
15m
Mitochondrial sub-fractions
Mitochondrial sub-fractions
10m
10m
Optional: To immunoprecipitate from crude mitochondria, first resuspend the mitochondrial pellet in TES buffer and sonicate for 1 min at 5s intervals (14uA amplitude) On ice
Incubate the crude mitochondrial fraction in ice cold digitonin (0.2 mg/mg mitochondrial protein) rolling or turning at 4 °C for 15 minutes.
9800 x g, 4°C, 00:10:00
10m
Pellet = mitoplast (IMM and matrix). Supernatant = OMM/IMS.
Optional: store supernatant or pellet resuspended in TES at -80 °C
Further fractionation
Further fractionation
1h
1h
Disrupt mitoplast by sonicating for 1 min at 5s intervals (14uA amplitude) On ice . Do the
same for the OMM/IMS fraction On ice .
Centrifuge all disrupted fractions: mitoplast, OMM/IMS and if needed cytosolic fraction at high speed. 100000 x g, 4°C, 01:00:00
1h
Mitoplast: Supernatant = matrix, pellet = IMM
OMM/IMS: Supernatant = IMS, pellet = OMM
Cytosol: Supernatant=purified cytosol, pellet = membrane contaminants.
Resuspend pellets in 100-200 µL TES and store all fractions at -80 C or use straight away.
Acknowledgements
Modified after a protocol developed for tissue by Prof. Ellen Billett (retired). Nottingham Trent University, UK