| | Re: Intact Mitochondria isolation
My apologies for the delay in my reply. Classes started this week, and I've been swamped prepping lectures and dealing with students.
Prepping mitochondria to high enough purity to easily find them in EM, while preserving their morphology, is about as difficult a task as you could have made for yourself. Nontheless, it can be done with practice.
The amount of starting material you are using should work, although you'd have better success for your application using a few grams of tissues. The protocol I used for EM, starting with about the same quantity of cells as you are:
1. Grow cells to confluence/saturation, isolate from plate/media as per usual method for cell type (i.e. trypsin/serum, scraping, etc).
2. Spin down cells 600g (1500rpm), 4oC, 10min.
3. Wash cells in 10ml MA buffer, spin down 600g (1500rpm), 4oC, 10min.
4. Suspend cells in 10ml MB buffer (make buffer fresh each time), put in a 50ml conical tube and load into ice-cold N2 cavitator or french press.
5. Pressurize N2 cavitator to 420PSI and let equilibrate for 20min. Collect sample drop-by-drop into a 50ml falcon tube. Or collect via usual french-press lysis method.
6. Add sucrose to a final concentration of 250mM (150μl/ml of 1M sucrose or 60μl of 2.5M sucrose) to restore normal osmolarity.
7. Transfer supernatant to 1.5ml epindorfs and spin at 2500g (5100RPM), 4oC, 5min. Transfer supernatant to clean epindorfs and repeat spin. Decant supernatant into clean epindorfs and spin down mitochondrial using a 10,000g (10,100RPM) spin, 15min, 4oC.
8. Resuspend pellet(s) in 1000μl of MC; if using more then 1 epindorf, serially reusspend pellet such that all mitochondria are suspended in the same 1000μl.
9. Wash 1-2x in MC buffer using a 10,000g (10,100RPM) spin, 15min, 4oC spin.
The above method will give you a crude extract. I'd do some EM on that first, to make sure your isolation is working, before moving onto the following steps. If the above is pure enough, USE IT. The remaining steps will improve your purity, but greatly reduce your yield.
1) Layer crude mitochondria isolate on top of 8ml percol isolation medium. If preparing from tissue prepare two tubes, placing 500μl of crude isolate ontop of each tube.
2) Balance tubes carefully, using addition of mitochondrial isolation buffer to mitochondrial layer.
3) Spin 95,000g, 30min, 4oC. Mitochondria will form a dense band ¾ of the way down – remove material above mitochondria first (contains peroxisomes and microscomes)
4) Dilute mitochondria with mitochondria isolation buffer, and spin down 6300g, 10min, 4oC. Repeat once.
5) Suspend final isolate in MSH Buffer without EDTA
MSH buffer: 10mM HEPES, 70mM Sucrose, 210mM Mannitol. pH to 7.35 with KHO.
MSH with EDTA: add 1mM EDT to MSH
Mito Isolation Buffer: 36ul/ml aprotinin (stock 1.8mg/ml), 5ul/ml PMSF (stock 200mM), 1ul/ml leupeptin (stock 10mM) in MSH without EDTA
Percol isolation medium: 39.3ml percoll, 73.5ml 10mM HEPES (pH 7.35), 13.2ml 2.5M sucrose.
MA: 100mM sucrose, 1mM EGTA, 20mM MOPS or 10mM HEPES (use MOPS or HEPES, not both). pH 7.4. Before use add 1g/l BSA.
MB: To MA without BSA add: 10mM triethanolamine, 5% percol
MC: To MA with BSA add: 200ul/ml sucrose