ISOLATION OF LARGE AMOUNTS OF HIGHLY PURE MITOCHONDRIA FOR "OMICS" STUDIES
Ultracentrifugation on a density gradient remains the only reliable method to obtain highly pure mitochondria preparations. However, it is not readily available for any laboratory and has a serious disadvantage of providing low mitochondria yield, which can be critical when working with limited starting material. Here we describe a combined method of mitochondria isolation for proteomic studies that includes cell disruption by sonication, differential cen-trifugation, and magnetic separation. Our method provides remarkable enrichment of mitochondrial proteins as compared to differential centrifugation, magnetic separation, or their combination, and it enables the strongest depletion of cytoplasmic components as assessed by two-dimensional electrophoresis, mass spectrometry, and Western blot. It also doubles the yield of mitochondria. However, our method should not be used for functional studies as most of the isolated organelles demonstrate disturbed structure on electron microphotographs.