Polysome Analysis and RNA Purification from Sucrose Gradients
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.
Key wordsTranslational control polysome profile RNA isolation sucrose gradient RNA denaturing electrophoresis
This work was supported by Czech Science Foundation grant No. 301/07/0607, Ministry of Education, Youth and Sports of the Czech Republic grant No. LC06066 (both to MP). LV was supported by The Wellcome Trusts grant No. 076456/Z/05/Z, Fellowship of Jan E. Purkyne from Academy of Sciences of the Czech Republic, and Inst. Research Concept AV0Z50200510.
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