A protocol for the subcellular fractionation of Saccharomyces cerevisiae using nitrogen cavitation and density gradient centrifugation.

ABSTRACT: Most protocols for yeast subcellular fractionation involve the use of mechanical shear forces to lyse the spheroplasts produced by the enzymatic digestion of the Saccharomyces cerevisiae cell wall. These mechanical homogenization procedures often involve the manual use of devices such as the Dounce homogenizer, and so are very operator-dependent and, in consequence, lack reproducibility. Here, we report a highly reproducible method of homogenizing yeast cells based on nitrogen cavitation. This has been optimized to allow efficient release of subcellular compartments that show a high degree of integrity. The protocol remains effective and reproducible across a range of sample volumes and buffer environments. The subsequent separation method, which employs both sucrose and iodixanol density gradients, has been developed to resolve the major membrane-bound compartments of S. cerevisiae. We present an integrated protocol that is fast, facile, robust and efficient and that will enable 'omics' studies of the subcellular compartments of S. cerevisiae and other yeasts.

SUBMITTER: Wang Y 

PROVIDER: S-EPMC4282465 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature