Isolating the Mitochondria
Introduction
Materials
- Ice bucket
- Cells
- Cell Culture Reagents
- RPMI
- PBS 1X
- Trypsin
- Mitochondria Isolation Kit Reagents
- Reagent A
- Reagent C
- TBS-CHAPS 2%
- Protease Inhibitor Cocktail, EDTA-Free from Pierce
- Microtubes (0.5mL & 2.0mL)
- Test Tubes (15mL)
- 1mL Syringe
- P-20 & P-1000
- Centrifuge
- Microcentrifuge
Procedure
Prepare Celll Suspension
- Equilibrate vial of Protease Inhibitor Cocktail to room temperature.
- Remove and discard the cell media from the bottle.
- Wash the cells with PBS 1X (25 cm2: 5mL, 75 cm2: 10mL & 150 cm2: 20mL) without Ca2+ & Mg2+, aspirate and discard the PBS from the bottle.
- Trypsinize cells
- Add Trypsin-EDTA (25 cm2: 0.5mL, 75 cm2: 2mL & 150 cm2: 4mL) to cover the cell layer.
- Place the bottle at 37ºC for 5 minutes; rock the bottle at 2 minutes intervals.
- Add RPMI-1640 complete medium to inactivate Trypsin (25 cm2: 4.5mL, 75 cm2: 8mL & 150 cm2: 6mL)
- Disaggregate cells by making them pass several times through the pipette against the bottom of the bottle.
- Transfer all cells to an appropriate 15mL test tube containing all the cells from the flask.
- Centrifuge the cells at 1200 rpm for 5 minutes.
- Remove and discard the medium.
- Resuspend the cells in X mL of medium, and wash the walls of the test tube with the medium to be sure to have all the cells in solution. X = (2mL) x (number of technical replicates to be made)
- Transfer the 2mL of cells to 2.0mL microtubes.
- Centrifuge the cells at 1,200 rpm for 5 minutes.
- Carefully remove and discard the supernatant.
- Put microtubes with pellet on ice.
Cell Lysis & Mitochondria Isolation
- Prepare 1mL of Mitochondrial Isolation Reagent A with protease inhibitors
- 1000 μL Mitochondrial Isolation Reagent A
- 10 μL Protease Inhibitor Cocktail for Vf = 10 μL/mL or 0.01 μL Inhibitor / 1 μL Lysis Buffer
Note: Briefly vortex Protease Inhibitor Cocktail vial immediately before use to ensure a homogeneous suspension of the cocktail components. For a higher potency protease inhibition, the Cocktail may be added to a 1.3X final concentration (e.g., 13 μL/mL).
- Add 800 μL of Mitochondrial Isolation Reagent A
- Moderately vortex for 5 seconds
- Incubate tube on ice for exactly 2 minutes.
- Transfer the cell suspension to 1mL syringe.
- Homogenize cells on ice. Perform ~80 - 100 strokes to effectively lyse the cells.
- Return lysed cells to original tube.
- Add 800 μL of Mitochondria Isolation Reagent C with protease inhibitor cocktail
- 800 μL Mitochondrial Isolation Reagent C
- 8 μL Protease Inhibitor Cocktail for Vf = 10 μL/mL or 0.01 μL Inhibitor / 1 μL Lysis Buffer
Note: Briefly vortex Protease Inhibitor Cocktail vial immediately before use to ensure a homogeneous suspension of the cocktail components. For a higher potency protease inhibition, the Cocktail may be added to a 1.3X final concentration (e.g., 13 μL/mL).
- Rinse the syringe with 200 μL of Mitochondria Isolation Reagent A and add to tube containing the sample.
- Invert tube several times to mix (DO NOT VORTEX).
- Centrifuge tube at 700 x g for 10 minutes at 4°C.
- Transfer the supernatant to a new microtube and discard the pellet.
- Centrifuge at 12,000 x g for 15 minutes at 4°C.
- Transfer the supernatant (CYTOSOL FRACTION) to a new tube label it and put in the ice.
CRITICALThe pellet contains the isolated mitochondria (DO NOT DISCARD IT).
- Prepare as many cytosolic fraction aliquots (100 μL) as possible. Store the samples at -20°C.
- Add 500 μL Mitochondria Isolation Reagent C to the pellet, and centrifuge at 12,000 x g for 5 minutes.
Discard the supernatant.
- CRITICALMaintain the mitochondrial pellet on ice before downstream processing.
Lysis of Mitochondria
- Prepare 100 μL of TBS-CHAPS 2% with 1μL of Pierce Protease Inhibitor Cocktail
- Add mix to the mitochondrial pellet.
- Vortex for 1 minute
- Centrifuge mitochondria at max speed for 2 minutes.
The supernatant contains soluble mitochondrial protein that can be analyzed by BCA Protein Assay.
- Make 3 aliquots of ~33 μL of the protein sample. Store the samples at -20°C.
Isolating the Mitochondria
Original protocol