[BioWHATT]

If a homogenate is centrifuged at 14 000 g for 10 minutes (4 degrees celsius) is it possible to extract mitochondria from the pellet?

[BioWHATT]

I centriguged a tissue homogenate at 14000 g for 10 minutes (4 degrees celsius).
How do I isolate mitochondria out of the resulting pellet or supernatant?

[Arkcon]

I've merged your two questions, because they are essentially the same.  I'm sorry you didn't get an answer the first time.  It's been a long time since my Mol Bio degree, and I didn't commit to memory the centrifugation rates for various organelles.  I even wonder if they're all the same, across various species -- maybe all animals are the same, or maybe not, maybe liver mitochondria are different from other organs, maybe plants are different from all others -- I can't be sure.  As I recall, in college, we just followed the recipe carefully, probably the same for industry.  So you may just want to check for a reference -- textbook, lab manual, class notes, random online cheat sheet, or whatever.  Unless you want to try varying speeds, to scout for all the g-force values for each organelle.  But, why would you do that?  Who's given you an organ lysate, a centrifuge, and said, "Go, figure it out."?

[BioWHATT]

Hi Arkcon

Thank you for the reply. I do have the correct method for rat brain mitochondria isolation (differential centrifugation). I just have a few samples that I started out with which I'd like to save. I began at high speed with those and now I would like to extract mitochondria out of the pellets if at all possible.

[Yggdrasil]

I would suggest looking up a reference such as Current Protocols in Cell Biology, which should have detailed procedures on how to isolate mitochondria.

[Babcock_Hall]

Terrance Cooper's book, The Tools of Biochemistry, has a section on isolating mitochondria from castor beans using two kinds of sucrose gradients (p. 347).  That may not be close enough to your situation, however.

[Arkcon]

If you think its likely you've pelleted out the mitochondria, along with other things at high speed, I suppose you could re-suspend in some sort of homogenizing solution, and try centrifuging again, using differential speeds a second time.  There's no reason not to try and salvage your experiment.

I remember my first such experiment, I was all set with a special centrifugation solution -- 0.25 M sucrose, NaCl, EDTA, Tris buffer at pH blah-blah.  I brought it to my adviser, a real old timer, and he asked, "You got the quarter molar sucrose ready?"  I explained what I'd made, and he was like, "fine, fine, as long as its cold and we work quickly."  Took me a couplea times doing this to learn this, yes the protocol is important, very likely the performance is improved by the chleator scavenging heavy metal ions, the salt keeping the environment natural, pH control is important.  But the real deciding factor in the art and science of molecular biology is cold, confidence, and speed.