Fig 1. Scheme of mitochondrial isolation from yeast using magnetic beads.

Yeast cells expressing Tom70-6xHis are converted to spheroplasts by incubation with Zymolyase, and disrupted using a Dounce homogenizer. The resulting whole-cell lysate is subjected to low-speed centrifugation to remove intact cells, cellular debris and nuclei, followed by high-speed centrifugation of the supernatant to concentrate mitochondria. We refer to the resuspended pellet from the high-speed centrifugation as the mitochondria-enriched fraction. The mitochondria-enriched fraction is the starting point for further purification of mitochondria. For affinity purification, the mitochondria-enriched fraction is incubated with HisPur Ni-NTA magnetic beads. Bead-bound mitochondria are separated from debris and other organelles in a magnetic field and eluted from beads with 500 mM imidazole.