Polysome analysis and RNA purification from sucrose gradients
Language English Country United States Media print
Document type Journal Article, Research Support, Non-U.S. Gov't
Grant support
076456
Wellcome Trust - United Kingdom
076456/Z/05/Z
Wellcome Trust - United Kingdom
- MeSH
- Centrifugation, Density Gradient methods MeSH
- Electrophoresis methods MeSH
- Data Interpretation, Statistical MeSH
- Yeasts MeSH
- Polyribosomes chemistry MeSH
- Protein Biosynthesis genetics MeSH
- Gene Expression Regulation genetics MeSH
- RNA isolation & purification MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- RNA MeSH
Velocity separation of translation complexes in linear sucrose gradients is the ultimate method for both analysis of the overall fitness of protein synthesis as well as for detailed investigation of physiological roles played by individual factors of the translational machinery. Polysome profile analysis is a frequently performed task in translational control research that not only enables direct monitoring of the efficiency of translation but can easily be extended with a wide range of downstream applications such as Northern and Western blotting, genome-wide microarray analysis or qRT-PCR. This chapter provides a basic overview of the polysome profile analysis technique and the RNA isolation procedure from sucrose gradients. We also discuss possible experimental pitfalls of data normalization, describe main alternatives of the basic protocol and outline a novel application of denaturing RNA electrophoresis in several steps of polysome profile analysis.
References provided by Crossref.org
Age-related differences in the translational landscape of mammalian oocytes
Distinct recruitment of human eIF4E isoforms to processing bodies and stress granules
