Schizosaccharomyces pombe Polysome Profile Analysis and RNA Purification
- 1Degenerative Disease Program, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California 92037;
- 2Department of Genetics, Evolution & Environment, and UCL Cancer Institute, University College London, London WC1E 6BT, United Kingdom;
- 3Center for RNA Molecular Biology and Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106-4906
- ↵4Correspondence: dwolf{at}sbpdiscovery.org; j.bahler{at}ucl.ac.uk
Abstract
Polysome profile analysis is widely used by investigators studying the mechanism and regulation of translation. The method described here uses high-velocity centrifugation of whole cell extracts on linear sucrose gradients to separate 40S and 60S ribosomal subunits from 80S monosomes and polysomes. Cycloheximide is included in the lysis buffer to “freeze” polysomes by blocking translation. After centrifugation, the gradient is fractionated and RNA (and/or protein) is prepared from each fraction for subsequent analysis of individual species using northern or western blots. The entire RNA population in each fraction can be analyzed by hybridization to microarrays or by high-throughput RNA sequencing, and the proteins present can be identified by mass spectrometry analysis.
Footnotes
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From the Fission Yeast collection, edited by Iain M. Hagan, Antony M. Carr, Agnes Grallert, and Paul Nurse.
