Intact Mitochondria isolation

[Nemesis05]

Hello apparently my lab have order too this kit: [Only registered users see links. ] .
I will test it apparently it's just a chemical homogenization.

[vigierex]

I really agree with the facts that you have shared on this post. An interesting topic like this really enhances reader's mind to have more effective decisions over a certain issue.

[Warthaug]

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

Solutions:
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

Bryan

[Nemesis05]

I have finally use this protcol last week with no success but i'm still working on to tune it and obtain better results. In fact with this protcol I obtained a lot of contamination with other things than mitochondria.
PREPARATION OF MITOCHONDRIA FROM CULTURED CELLS

Prepare cells

1. Wash cultures twice with PBS

2. Trypsinize the cells and pellet it at 1000 g, room temperature for 15 min.

3. Aspirate all of the supernatant and resuspend the cells in ice-cold cell
homogenization medium. Use a volume of medium equal to six times the volume of the pellet.
4. Leave on ice for 2 min.

Homogenize cells

5. use cold Potter-Elvehjem homogenizer

6. Add 1⁄3 vol ice-cold CHM containing 1 M sucrose (final 0.25 M) and mix gently by repeated inversion.

Isolate mitochondria

7. Pellet nuclei by centrifuging 5min at1000,4°C.

8. aspirate the supernatant and centrifuge 10 min at 5000g, 4°C.

9. Resuspend the pellet in 10 ml ice-cold sucrose/Mg2+ medium using two to three gentle strokes of the pestle in a Dounce homogenizer.

10. Recentrifuge at 5000 g, 10 min, 4°C. Resuspend the pellet in 2 to 3 ml ice-cold mitochondrial suspension medium I

REAGENTS AND SOLUTIONS

Cell homogenization medium (CHM)

To 100 ml H2O add:
30 μl 1 M MgCl2 (150 mM final)
0.15 g KCl (10 mM final)
2.0 ml 1 M Tris%Cl Adjust pH to 6.7
Add H2O to 200 ml
For CHM containing 1 M sucrose, add 68.4 g sucrose to 200 ml CHM.

Mitochondrial suspension medium I

To 50 ml H2O add:
8.5 g sucrose (0.25 M final) 1.0 ml 1 M Tris base (10 mM final) Adjust pH to 7.0 with acetic acid Add H2O to 100 ml Store up to 1 to 2 days at 4°C

Sucrose/Mg2+ medium

To 100 ml H2O add:
30 μl 1 M MgCl2 (0.15 M final) 17.1 g sucrose (0.25 M final) 2 ml 1 M Tris%Cl (APPENDIX 2A; 10 mM final) Adjust pH to 6.7 Add H2O to 200 ml Store up to 1 to 2 days at 4°C.

Thanks a lot for your protocol I will try it soon, when you say you check by em your crude extract of mitochondria, (do you do negative stain or section).
Thanks for your help.
I will letsyou know what is finally work for my case

thanks

[Nemesis05]

I want to add a precision, I have replaced the sucrose by KCL in the final buffer Mitochondrial suspension medium I

To 50 ml H2O add:
KCL 0.15 M final1.0 ml 1 M Tris base (10 mM final) Adjust pH to 7.0 with acetic acid Add H2O to 100 ml Store up to 1 to 2 days at 4°C. Sugar are not really good for EM studies

Do you know a technique to check the presence of mitochondir in my sample before I use it for EM. ( fluorecencse , optical microscopy, ...) I'll planned to use the mito tracker dye on an aliquots of my sample and check by concfocal microscopy.

[Nemesis05]

I'm finally really frustrated, ihave tired all the differents protocols mentioned above (pierce, myletinyl biotech, the Obama one and the last on that I have posted last time) and on the EM images I saw a lot of things except Mitochondria. I saw a lot of liposomes or other things. Do you know what it's wrong with my purification. I will try different things in the future like increase edta or egta concentration, follow the lysis of cells, increase the number of cells, etc....
Here you can see an example of what i can obtained
http://imageshack.us/photo/my-images...ositive1f.jpg/

thanks in advance

[Nemesis05]

I have found this interesting paper
Comparative analyses of cell disruption methods for mitochondrial isolation in high-throughput proteomics study
[Only registered users see links. ]

thanks

[Nemesis05]

I have tried to post several messages to answer and thank you, but a least only one messages appeared.
I have tried pierce, myletynil; PREPARATION OF MITOCHONDRIA FROM CULTURED CELLS and obama's protocol. I obtained every time the same kind of results ( only a lot of vesicules and really few dead mitochondria) . I think it's really due to the way to brake the cells and maybe the quantity of cells. I found this paper (Comparative analyses of cell disruption methods for mitochondrial isolation in high-throughput proteomics study
Sakdithep Chaiyarit a,b, Visith Thongboonkerd a,)
and I will try the sonication what do you think about that.
Thanks

[Nemesis05]

Here you can see some EM images of my results

By [Only registered users see links. ] at 2011-09-19

[protolder]

Hola I have found this protocol for other forer: [Only registered users see links. ]. I´m not sure if it isolate intact mitochondia or separate their proteins. Buena suerte