Purification of 70S ribosomes

Maria C Rivera; Bruce Maguire; James A Lake

doi:10.1101/pdb.prot081356

Purification of 70S ribosomes

Cold Spring Harb Protoc. 2015 Mar 2;2015(3):300-2. doi: 10.1101/pdb.prot081356.

Authors

Maria C Rivera¹, Bruce Maguire², James A Lake³

Affiliations

¹ Department of Biology and Center for the Study of Biological Complexity, Virginia Commonwealth University, Richmond, Virginia 23284;
² Primary Pharmacology Group, Pfizer Global Research and Development, Groton, Connecticut 06340;
³ Department of Molecular, Cell and Developmental Biology and Department of Human Genetics, University of California, Los Angeles, California 90095.

PMID: 25734066
DOI: 10.1101/pdb.prot081356

Abstract

Here we describe the further purification of prokaryotic ribosomal particles obtained after the centrifugation of a crude cell lysate through a sucrose cushion. In this final purification step, a fraction containing ribosomes, ribosomal subunits, and polysomes is centrifuged through a 7%-30% (w/w) linear sucrose gradient to isolate tight couple 70S ribosomes, as well as dissociated 30S and 50S subunits. The tight couples fraction, or translationally active ribosome fraction, is composed of intact vacant ribosomes that can be used in cell-free translation systems.

MeSH terms

Centrifugation, Density Gradient / methods*
Complex Mixtures
Prokaryotic Cells / chemistry
Ribosomes*

Substances

Complex Mixtures