Hi...I am trying to isolate mitochondria from cultured mammalian cells using OptiPrep density gradient. However, in the literature I found that Percoll has been widely used with the best separation between mitochondria and ER. Can someone suggest me the intricacies of the methods so that I can obtain optimal separation between mitochondria and ER using Optiprep as I want best purity of mitochondria with least contamination of ER?
It is hard to recommend with no name of the cells that you are going to use. For some cells/mitochondria Percoll is better. Please see the paper attached with the method for neuronal mitochondria. To get best results you might need to adjust/improve the method/protocol that was used by other people. that is what research is about. Percoll was and is widely used for gradient organelle separation. However, the contamination of the mitochondrial fraction by ER fraction is possible. You have to run separate western blots with specific markers for mitochondria and for ER (like GRP78) to make sure that fraction is clean. It depends on the aim of your experiment - some contamination might be hard to avoid, but they are not necessarily going to influence your results.
Thank you Olga for a detailed discussion. I am working with HeLa cells. I have seen most papers where there is absolutely no ER contamination in the mitochondrial fraction with Percoll preparation. The paper you recommended uses Percoll fractionation together with ani-TOMM20 dependent purification together. I am getting substantial ER contamination in my mitochondrial fraction obtained using Opti-Prep gradient as confirmed by western blotting for ER marker (Calnexin) and Mitochondrial marker (VDAC). My experimental requirements does not tolerate significant ER contamination.
Optiprep (60%w/v) has a max density of 1.32 g/ml while Percoll is 1.13 g/ml. When you modify this with your buffer system you will have a much steeper gradient form with optiprep. The net result will be to move "peaks" of material of similar density closer together. Why not just use percoll? It appears from your results that Optiprep does not work as well.
On the other hand, can you use Percoll to substitute Optiprep if you can only one of these solutions? I want to isolate some cells from spinal cord tissue, one protocol uses Optiprep and another used Percoll. I'm conflicted as to which one I should be using here. Olga Sukocheva Peter Pediaditakis
Yun Li
If only one, then Optiprep. Lower osmolality (260 mOsm) at the supplied density (1.32) means that it will have broader applications where lower osmolality is needed ( sperm).
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