Volume 703 of the series Methods in Molecular Biology pp 293-309


Polysome Analysis and RNA Purification from Sucrose Gradients

  • Tomáš MašekAffiliated withDepartment of Genetics and Microbiology, Charles University in Prague Email author 
  • , Leoš ValášekAffiliated withLaboratory of Regulation of Gene Expression, Institute of Microbiology, Academy of Sciences of the Czech Republic
  • , Martin PospíšekAffiliated withDepartment of Genetics and Microbiology, Faculty of Science, Charles University in Prague

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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 words

Translational control polysome profile RNA isolation sucrose gradient RNA denaturing electrophoresis