Abstract
This unit presents detailed protocols for a range of centrifugation-based subcellular fractionation procedures for the yeast Saccharomyces cerevisiae. Techniques include spheroplast preparation, glass-bead lysis, differential centrifugation, and several density gradient procedures using a variety of gradient media. There are analytical procedures that are primarily designed to evaluate the association of proteins with organelles in the exocytic and endocytic pathways. Additionally, there are preparative protocols for isolation of yeast nuclei, vacuoles, mitochondria, peroxisomes, endoplasmic reticulum, plasma membrane, and cytosol. The unit also contains a table, with references, for alternative approaches to isolation of these organelles and fractions.
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