Isolation of highly purified mitochondria from Saccharomyces cerevisiae.

摘要: Publisher Summary This chapter focuses on the isolation of highly purified mitochondria from Saccharomyces cerevisiae and purification a 10-liter culture wild-type yeast cells, grown in stirred-tank fermenter. A typical yield is 50 to 100 mg mitochondrial protein. The procedure can be scaled up or down as appropriate. For example, 20 25 protein obtained four l-liter cultures Erlenmeyer flasks with vigorous aeration. Isolated fixed then processed for thin-section electron microscopy. Fixation achieved simply by adding glutaraldehyde concentrated stock solution. However, two modifications this method facilitate subsequent analysis. First, it often desirable shrink inner membrane–matrix compartment placing hypertonic approach gives an indication intactness organelles, because will only if membrane osmotically tight. In shrunken mitochondria, contact sites between membranes are more easily visualized. Second, addition tannic acid during fixation greatly enhances contrast, particularly structures.