anyone gives me some tips on mitochondria isolation??
Shu Liu
Wednesday, 13 May 2009 14:46 UTC
I’m working on a certain protein which locate both in the cytosol and mitochondria intermembrane space. well, I just want to test the mitochondrial pool of this protein, therefore I need to isolate mitochondria with the minimal contamination of cytosolic fraction. has anyone ever done this kind of experiments?
To lysis cells, digitonin solution and dounce homogenizer, which one is better?
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Replies
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Check this Paper.
It has a wonderful protocol. For more details you can mail the authors
(pfigueiredo@ismai.pt)
Aging Impairs Skeletal Muscle Mitochondrial
Bioenergetic FunctionPedro A. Figueiredo , 1 Scott K. Powers , 2 Rita M. Ferreira , 1 Hans Joachim Appell , 1 , 3 and José A. Duarte 1
Journal of Gerontology: BIOLOGICAL SCIENCES
2009. Vol. 64A, No. 1, 21 – 33 -
The way to isolate mitochondrion depends on about what do you want to assay exactly. If you need a functional mitochondrion with high integrity and RCR I recomend you dounce homogenizer to avoid any damage in the external membrane. Wich tissue are the mitochondrion from? The protocol(gs and isolation medium) could vary change in function of the tissue.
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Thank you guys so much~
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Shu,
If you need to mechanically disrupt the cells to preserve the mitochondrial membrane and you would like a faster or higher-throughput method than homogenizing with a dounce by hand, you may want to consider a mechanical homogenizer. My company sells one – the Bullet Blender – which is very cheap compared to other similar units and is very effective as well. It would also give you far more consistency in your results than would any manual method like a dounce. It may not be worth it if you’re only doing two or three samples at a time, or you will only be performing homogenizations for a brief while, but over time a homogenizer would save you a whole lot of time.
If I can be of any help, let me know.
Cheers,
-Carlton
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