用于制备应用的过氧化物酶体的大规模纯化

Large-Scale Purification of Peroxisomes for Preparative Applications.

作者信息

Cramer Jana, Effelsberg Daniel, Girzalsky Wolfgang, Erdmann Ralf

机构信息

Department of System Biochemistry, Institute of Biochemistry and Pathobiochemistry, Medical Faculty, Ruhr-University Bochum, D-44780 Bochum, Germany.

出版信息

Cold Spring Harb Protoc. 2015 Sep 1;2015(9):pdb.prot083725. doi: 10.1101/pdb.prot083725.

DOI:10.1101/pdb.prot083725

PMID:26330621

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.

摘要

本方案旨在通过连续两次密度梯度离心从酿酒酵母中大规模分离高纯度过氧化物酶体。提供了收获多达60克油酸诱导的酵母细胞以制备原生质球并生成富含过氧化物酶体和线粒体的细胞器沉淀（OPs）的操作说明。将OPs加载到八个连续的36%-68%（w/v）蔗糖梯度上。离心后，通过测定过氧化氢酶活性来确定过氧化物酶体的峰值部分。随后将这些部分合并，并使用20%-40%（w/v）的Nycodenz进行第二次密度梯度离心。