Large-Scale Purification of Peroxisomes for Preparative Applications
- Department of System Biochemistry, Institute of Biochemistry and Pathobiochemistry, Medical Faculty, Ruhr-University Bochum, D-44780 Bochum, Germany
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.
Footnotes
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↵1 Correspondence: ralf.erdmann{at}rub.de
- © 2015 Cold Spring Harbor Laboratory Press
