Obtaining Mitochondrial Fractions - Possibly Stupid Question (Jul/21/2008 )
My prof wants some mitochondria to get some quick data on mitochondrial membranes for a grant proposal he's writing. I have a few concerns on this, the most important is on how important it is to have fresh liver or other tissue to harvest mitochondria from? The protocols I can find involve harvesting the liver from rats (which we currently don't have) and then immediately starting the harvesting protocol.
Secondly, while we currently don't have access to purpose-grown animals, I do work for a large university with a livestock program. Would harvesting mitochondria from a liver section of a larger mammal raise any additional issues? Also, how quickly would I have to harvest the mitochondria after obtaining the liver section?
You want to isolate your mitochondria just after dissecting your tissue, as freezing/thawing will make them burst and therefore you won't be able to isolate them. How fast you will have to isolate them will depend on what is it that you want to do with them, i.e. if you want to look at enzymatic activities (some of them can get inactivated quite quickly), the sooner the better, if you want to look at protein expression, timing won't be as crucial (although of course you'll be dealing with protein degradation too). It's important to do the whole procedure keeping the sample always on ice...
Hope this helps