核糖体和多核糖体的分离

Isolation of ribosomes and polysomes.

作者信息

Rivera Maria C, Maguire Bruce, Lake James A

机构信息

Department of Biology and Center for the Study of Biological Complexity, Virginia Commonwealth University, Richmond, Virginia 23284;

Primary Pharmacology Group, Pfizer Global Research and Development, Groton, Connecticut 06340;

出版信息

Cold Spring Harb Protoc. 2015 Mar 2;2015(3):293-9. doi: 10.1101/pdb.prot081331.

DOI:10.1101/pdb.prot081331

PMID:25734065

Abstract

Here we describe a preparative differential centrifugation protocol for the isolation of ribosomes from a crude cell homogenate. The subcellular fraction obtained is enriched in ribosome monomers and polysomes. The protocol has been optimized for the homogenization and collection of the ribosomal fraction from prokaryotic cells, mammalian and plant tissues, reticulocytes, and chloroplasts. The quality of the ribosomal preparation is enhanced by the removal of the remaining cellular components and adsorbed proteins by pelleting through a sucrose cushion with a high concentration of monovalent salts, NH4Cl or KCl. The different components of the ribosomal fraction isolated using this protocol can be further purified by sucrose gradient centrifugation.

摘要

在此，我们描述了一种用于从粗细胞匀浆中分离核糖体的制备性差速离心方案。所获得的亚细胞级分富含核糖体单体和多聚核糖体。该方案已针对从原核细胞、哺乳动物和植物组织、网织红细胞及叶绿体中匀浆化和收集核糖体级分进行了优化。通过在高浓度单价盐（NH4Cl或KCl）存在下经蔗糖垫层离心沉淀以去除剩余细胞成分和吸附蛋白，可提高核糖体制备物的质量。使用该方案分离得到的核糖体级分的不同组分可通过蔗糖梯度离心进一步纯化。