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Nemesis05 08-29-2011 03:10 PM

Intact Mitochondria isolation
 
I'm looking for a good protocol to purify intact mitochondria from cultured cells.
I have used a lot of different kit, but the dounce homgenization part is to crtical, and I obtained a lot of broken mitochondria and the sample was not really pure.
I'll planed to use the pierce kit

Have you some good advices for me.


thanks

Warthaug 08-31-2011 06:00 PM

Re: Intact Mitochondria isolation
 
I've used the kits - they suck; save your money.

Isolating mitochondria is not trivial, and I need to know more before I recommend a protocol. Please answer the following questions:
1) What cell type are you isolating from?
2) How many cells (or what sized plate) are you preforming the isolation from?
3) How pure do you need them to be?
4) What is the intended use (i.e. what quantities do you need, any how healthy do they need to remain.

Bryan

Nemesis05 09-01-2011 06:51 AM

Re: Intact Mitochondria isolation
 
Thanks for you answer

1) What cell type are you isolating from?
Hela cells
Cos 1
CHO
2) How many cells (or what sized plate) are you preforming the isolation from?
5 petri dishes (5x 150mm plates of cells)
3) How pure do you need them to be?
as much as possible
4) What is the intended use (i.e. what quantities do you need, any how healthy do they need to remain.
it's for em 25 uL will be sufficient and healthy as well too.

thanks

I will try the classical differential centrifugation protocols.

thanks again
Bryan

Nemesis05 09-01-2011 01:23 PM

Re: Intact Mitochondria isolation
 
I would say 5 petri dishes 25 150 mm 90 % confluency

sorry

nemesis

Nemesis05 09-02-2011 07:34 AM

Re: Intact Mitochondria isolation
 
I would mean 5 petri dishes (25x 150mm plates of cells)
sorry
I really need to finalize this purification to have a good start for my post doc.

thanks

obama 09-02-2011 12:05 PM

Re: Intact Mitochondria isolation
 
Initially i used mitochondrial isolation kit. But i got better result when i used our homemade protocols and buffer. If you interest let me know (i have to look for it in my lab note).

And it will good if you write, what is the end use of this mitochondria. mean what kind of experiment you are going to perform with this mitochondria.

Nemesis05 09-02-2011 12:31 PM

Re: Intact Mitochondria isolation
 
Thanks for you answer

I have already mentioned my goal
My intended use is Electron microscopy analysis of mitochondria parts
Yes I'm very interest by your homemade protocol
What kind of kit have you already use
I have use with any success the QIAGEN and miltenyibiotec kits
thanks for your help

obama 09-02-2011 01:46 PM

Re: Intact Mitochondria isolation
 
I used Qiagen before

Here is brief protocol

1. cells were permeabilized by resuspension in buffer containing 210 mmol/liter sucrose, 20 mmol/liter HEPES-KOH (pH 7.5), 10 mmol/liter KCl, 1.5 mmol/liter MgCl2, 1.0 mmol/liter EDTA, 1.0 mmol/liter EGTA, 1.0 mmol/liter dithiothreitol, 0.1 mmol/liter phenylmethylsulfonyl fluoride, 1:100 protease inhibitor mixture, and 0.04% digitonin.

2. Keep it for 45 min in ice (in between mix it) and followed homogenization using Dounce homogenization procedure it look like this (do five times up and down ) [Only registered and activated users can see links. Click Here To Register...]

3. i also passed the solution using 21gugage syringe to further homogenize it.

4. Then sediment nuclei and cellular debris, cell suspensions were centrifuged at 600 g for 10 min at 4 C. The supernatant was centrifuged at 15,000 g for 30 min at 4 C.

5. After this centrification, the pellet was defined as the mitochondrial fraction.

I used this mitochondria for western blot to analysis protein trans-location experiment. but you can try it.

Nemesis05 09-05-2011 08:38 AM

Re: Intact Mitochondria isolation
 
Ok I will try taht soon
Thanks

Nemesis05 09-05-2011 08:59 AM

Re: Intact Mitochondria isolation
 
I found a protocol which is adapted from Attardi and Ching (1979) and Rice and Lindsay (1997), is designed for cultured cells (2.10^8) grown as a monolayer. It's really close to your protocol. I hope it will be enough pure for my EM experiement
thanks


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