Large-Scale Purification of Peroxisomes for Preparative Applications.
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.
Department of System Biochemistry, Institute of Biochemistry and Pathobiochemistry, Medical Faculty, Ruhr-University Bochum, D-44780 Bochum, Germany.
Jana
J
Cramer
Department of System Biochemistry, Institute of Biochemistry and Pathobiochemistry, Medical Faculty, Ruhr-University Bochum, D-44780 Bochum, Germany.
Daniel
D
Effelsberg
Department of System Biochemistry, Institute of Biochemistry and Pathobiochemistry, Medical Faculty, Ruhr-University Bochum, D-44780 Bochum, Germany.
Wolfgang
W
Girzalsky
Department of System Biochemistry, Institute of Biochemistry and Pathobiochemistry, Medical Faculty, Ruhr-University Bochum, D-44780 Bochum, Germany.
Ralf
R
Erdmann
D002374:Catalase / Q000032:analysis; D002458:Cell Fractionation / Q000379:methods*; D002499:Centrifugation, Density Gradient / Q000379:methods*; D045424:Complex Mixtures; D020675:Peroxisomes; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000201:enzymology*; D013104:Spheroplasts / Q000737:chemistry / Q000201:enzymology
D045424:Complex Mixtures; D002374:Catalase