Hi everyone,
Does anyone know a really working mitochondrial isolation protocol? I need a quick separation method for a large number of cells. A quick protocol is needed because the substances that I am planning to determine are unstable.
See it this helps: Frezza C, Cipolat S, Scorrano L (2007) Organelle isolation: Functional mitochondria from mouse liver, muscle and cultured filroblasts. Nat Protoc 2:287–295. https://doi.org/10.1038/nprot.2006.478
Seahorse protocols worked well for me, be it cell pellets or mouse/rat liver. A good example can be also found here:
http://www.ruf.rice.edu/~bioslabs/studies/mitochondria/mitoprep.html
Human/rat/mouse tissue was minced and homogenized in isolation buffer [320 mM sucrose, 5 mM Tris (hydroxymethyl) methylaminoethane sulfonic acid (TES), 1 mM EGTA, pH 7.2]. The homogenate was then centrifuged at 1000 g for 5 min at 4 °C. The supernatant was collected and the pellet was resuspended in isolation buffer followed by second round of homogenization and centrifugation as mentioned earlier. The supernatants from both the steps were then pooled and centrifuged at 8,500 g for 10 min at 4°C. The crude mitochondrial fraction obtained in the pellet was resuspended in minimum volume of isolation buffer (~200 µl), overlaid on 6 % (w/v) ficoll solution, and centrifuged at 75,000 g for 30 min at 4°C, to remove the myelin content. The pellet was then resuspended in ~150 µl reconstitution buffer (250 mM sucrose, 10 mM TES, pH 7.2) and stored at -80 °C.
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