I used to make mitochondria a few decades ago on a routine basis so I can give you some general idea but not specific protocols to follow to a letter.

I think that you should be able to find many detailed protocols from literature searches.

Key aspects: Everything must be pre-chilled to 0ºC

1. Homogenization solution should be either 0.25 Sucrose or Mannitol

2. Start with very fresh liver (bleed the animal as best as possible to remove RBC from liver).

3. Use loose fitting teflon/glass homogenizer (pre chilled, of course) at relatively low rotational speed (my guess is that no more than 400RPM) with only as many up/down strokes as minimally needed for even suspension of tissue in 3 vol of Sucrose/Mannitol. While homogenizing, hold the homogenizer in a beaker with ice to ensure that temp, due to friction, does not rise.

4. The first spin to remove cells, debris, nuclei etc is at 600 x g for 10min. Save the supernatant in ice bucket.

5. Add 1/4th or 1/3rd the vol of sucrose (compared to the first time), mix carefully to resuspend (no vortexing), spin at 600xg for 10 min and combine the two supernatants.

6. Combined 600xg Supt is centrifuged at 15000xg for 5 min.

7. Discard the supernatant - save the loosely packed pellet.

8. Wash the mitochondria 2 times by re-suspending and spinning at 15,000xg. This step will reduce the microsomal fraction considerably (pinkish as opposed to buff/brownish mito fraction).

Since you have the maximum speed of 12,000g only, you may loose some mitochondria (smaller ones) or you may have to increase time a little more than 5 min. But try for 5 min first, if near-quantitive recovery is not your goal, you may be pleasantly surprised how much you will collect. Under EM, a large majority of mitochondria collected in this manner are coupled with condensed christae.

[PS: Disclaimer - I may have forgotten some details after over 3 decades; do please check something like the appropriate volume of Methods in Enzymology (Vol: 10?? - Author: Lardy) etc. Of corse, all glassware should be meticulously cleaned (I used to use an overnight Chromic acid soak) and solutions should be free of dissolved CO2 for high quality mitochondria. 

"can only run at room temperature (ie 4 degrees) and 12,000g"

The speed is fine but the temp could be a problem.

I assume that you have a cold room. in which case, you may be alright - 

It depends on what is it you wish to do with mitochondrial fraction - if your work requires actively respiring organelle (condensed christae), i expect that results will not be to your liking.

If on the other hand, by room temp you actually mean ~20ºC, the quality may be worse still.

Hi sorry there was a typo there. I meant we can't run at 4 degrees. But only room temp. We don't have a cold room in cat 3. Everything generally has to be at to temp but I want to isolate the mitochondrial fraction for western blotting.  Does this help now I have written the right temp? 

I shouldn't think so. During the run, the sample temperature raises significantly, so it must be done in a cold room at about 4-5° C at the most. You may have buffers and samples on ice for some time (1h?) before the operation,but yours results may not be all coherent. Alterations in the structure of the proteins during the preparation could complicate your data. Sorry! 

I agree with Joao and Tausif. Mitochondrial isolation requires you to keep the samples carefully chilled. I have seen that some labs utilize a (table-top) centrifuge in a large fridge. Maybe you could do something similar?

Cheers, Christian

Sathyavani,

If you have no choice, give it a whirl with what you have. Depending on the protein(s) of your interest, you may or many not see much of a difference on a Western blot - not all proteins are equally sensitive to degradation. Once you have the mitochondrial pellet, resuspend it in a chilled buffer with protease inhibitors and keep it on ice while you make protein measurements (if necessary) and treat the sample with SDS/DTT (BME) in boilng water as quickly as you can. 

All concerns and precautions are far more important if you were studying functional activity of mitochondria or its components.

Best,

Thanks so much to all - I am sure this advice might be useful for others in a similar predicament.  I am aware that some rotas and bench centruifuges can be put inside the fridge but we have an older one which wouldn't allow that. Tausif I shall perhaps try the isolation when I am ready  - do you have a protocol that you might recommend at all?  Best wishes, Vani 

I used to make mitochondria a few decades ago on a routine basis so I can give you some general idea but not specific protocols to follow to a letter.

I think that you should be able to find many detailed protocols from literature searches.

Key aspects: Everything must be pre-chilled to 0ºC

1. Homogenization solution should be either 0.25 Sucrose or Mannitol

2. Start with very fresh liver (bleed the animal as best as possible to remove RBC from liver).

3. Use loose fitting teflon/glass homogenizer (pre chilled, of course) at relatively low rotational speed (my guess is that no more than 400RPM) with only as many up/down strokes as minimally needed for even suspension of tissue in 3 vol of Sucrose/Mannitol. While homogenizing, hold the homogenizer in a beaker with ice to ensure that temp, due to friction, does not rise.

4. The first spin to remove cells, debris, nuclei etc is at 600 x g for 10min. Save the supernatant in ice bucket.

5. Add 1/4th or 1/3rd the vol of sucrose (compared to the first time), mix carefully to resuspend (no vortexing), spin at 600xg for 10 min and combine the two supernatants.

6. Combined 600xg Supt is centrifuged at 15000xg for 5 min.

7. Discard the supernatant - save the loosely packed pellet.

8. Wash the mitochondria 2 times by re-suspending and spinning at 15,000xg. This step will reduce the microsomal fraction considerably (pinkish as opposed to buff/brownish mito fraction).

Since you have the maximum speed of 12,000g only, you may loose some mitochondria (smaller ones) or you may have to increase time a little more than 5 min. But try for 5 min first, if near-quantitive recovery is not your goal, you may be pleasantly surprised how much you will collect. Under EM, a large majority of mitochondria collected in this manner are coupled with condensed christae.

[PS: Disclaimer - I may have forgotten some details after over 3 decades; do please check something like the appropriate volume of Methods in Enzymology (Vol: 10?? - Author: Lardy) etc. Of corse, all glassware should be meticulously cleaned (I used to use an overnight Chromic acid soak) and solutions should be free of dissolved CO2 for high quality mitochondria. 

You cannot isolate mitochondria at room temperature, you need cild centrifuge 0-4 Degrees Centigrade. Also, your table to will generate frictional heat at 12000xg which will further ruin your preparation.

I would be careful choosing the protocol for isolation. Specifically adopting a protocol for  liver mitochondria isolation to isolation from cells.

Aside of differential centrifugation why not use a different separation technique? Say, magnetic beads with immobilized anti-mitochondrial antibodies. In this case you would need a centrifugation to be the last and very quick step (~5 min at 8000xg)