Isolation of Highly Purified, Intact, and Functional Mitochondria from Potato Tubers Using a Two-in-One Percoll Density Gradient

The isolation of mitochondria from potato tubers (Solanum tuberosum L.) is described, but the methodology can easily be adapted to other storage tissues. After homogenization of the tissue, filtration and differential centrifugation, the key step is a Percoll density gradient centrifugation. The Percoll gradient contains two parts: a bottom part containing Percoll in 0.3 M sucrose, and a slightly less dense top part containing Percoll in 0.3 M mannitol. After centrifugation, a density gradient is formed that is almost linear in the central part, and this is where the band containing the purified intact mitochondria is formed. This method makes it possible to process large amounts of plant material (2-6 kg) and saves at least 1.5 h on the preparation time compared to methods where two consecutive purification methods are used. Nonetheless, it yields large amounts of mitochondria (50-125 mg protein) of very high purity, intactness and functionality.