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Purification of Saccharomyces cerevisiae Mitochondria by Zone Electrophoresis in a Free Flow Device

2025-03-29 生物化学 加入收藏
This chapter describes the isolation of yeast mitochondria by differential centr

This chapter describes the isolation of yeast mitochondria by differential centrifugation followed by mitochondrial purification through zone electrophoresis (ZE) using a free flow device (FFE). Starting from a yeast colony, cultures are grown under respiratory conditions to logarithmic phase. Cells are collected, their cell walls enzymatically disintegrated and the resulting spheroplasts are homogenized. Mitochondria are pre-fractionated from this homogenate by differential centrifugation. With the focus on further purification, pre-fractionated mitochondria are subjected to ZE-FFE. In ZE-FFE, mitochondria are transported with the buffer flow through the separation chamber and purified from contaminants by specific deflection through a perpendicularly oriented electric field. The purified mitochondria can be collected by centrifugation and used for further experiments and analysis such as sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE), immunoblotting, 2-DE or electron microscopy.

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