Large-Scale Purification of Peroxisomes for Preparative Applications

Abstract

This protocol is designed for large-scale isolation of highly purified peroxisomes from Saccharomyces cerevisiae using two consecutive density gradient centrifugations. Instructions are provided for harvesting up to 60 g of oleic acid-induced yeast cells for the preparation of spheroplasts and generation of organellar pellets (OPs) enriched in peroxisomes and mitochondria. The OPs are loaded onto eight continuous 36%-68% (w/v) sucrose gradients. After centrifugation, the peak peroxisomal fractions are determined by measurement of catalase activity. These fractions are subsequently pooled and subjected to a second density gradient centrifugation using 20%-40% (w/v) Nycodenz.

Analysis of Yeast Peroxisomes via Spatial Proteomics

“…Measure the OD600 as described in step 4 to determine the efficiency of cell wall digestion. For sufficient digestion, the OD600 after incubation should be ≤ 20% of the value determined before addition of DTT and Zymolyase 20-T [40].…”