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Isolation and Analysis of Bacterial Ribosomes Through Sucrose Gradient Ultracentrifugation

Part of the Methods in Molecular Biology book series (MIMB,volume 2106)

Abstract

Ribosomes are large macromolecular complexes responsible for the translation process. During the course of ribosome biogenesis and protein synthesis, extra-ribosomal factors interact with the ribosome or its subunits to assist in these vital processes. Here we describe a method to isolate and analyze not only bacterial ribosomes but also their associated factors, providing insights into translation regulation. This detailed protocol allows the separation and monitoring of the ribosomal species and their interacting partners along a sucrose density gradient. Simultaneously, fractionation of the gradient allows for the recovery of 70S ribosomes and its subunits enabling a wide range of downstream applications. This protocol can be easily adapted to ribosome-related studies in other species or for separating other macromolecular complexes.

Key words

  • Ribosomes
  • Ribosome isolation
  • Ribosome profiles
  • Sucrose gradient
  • Ultracentrifugation
  • UV analysis

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  • DOI: 10.1007/978-1-0716-0231-7_19
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Change history

  • 24 March 2020

    This chapter was inadvertently published without including the author Cátia Bárria. The correct authorship for this chapter should have been Ricardo F. dos Santos, Cátia Bárria, Cecília M. Arraiano, and José M. Andrade. And the sentence before the final sentence in the acknowledgement section should have been printed as “R.F.dS. is recipient of an FCT Doctoral fellowship (PD/BD/105733/2014) and Cátia Bárria is recipient of a FCT Post-doctoral grant PTDC/BIA-BQM/28479/2017)”. These corrections have been updated in the chapter.

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Acknowledgments

This work was financially supported by Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER through COMPETE2020-Programa Operacional Competitividade e Internacionalização (POCI) and by FCT-Fundação para a Ciência e a Tecnologia (Portugal), including Program IF (IF/00961/2014) and Grants PTDC/BIA-MIC/32525/2017 to J.M.A. and PTDC/BIA-MIC/1399/2014 to CMA; R.F.dS. is recipient of an FCT Doctoral fellowship (PD/BD/105733/2014) and Cátia Bárria is recipient of a FCT Post-doctoral grant PTDC/BIA-BQM/28479/2017). We also acknowledge the European Union Horizon 2020 Research and Innovation Programme grant agreement no. 635536 to CMA.

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Correspondence to Cecília M. Arraiano or José M. Andrade .

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dos Santos, R.F., Bárria, C., Arraiano, C.M., Andrade, J.M. (2020). Isolation and Analysis of Bacterial Ribosomes Through Sucrose Gradient Ultracentrifugation. In: Heise, T. (eds) RNA Chaperones. Methods in Molecular Biology, vol 2106. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0231-7_19

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  • DOI: https://doi.org/10.1007/978-1-0716-0231-7_19

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0230-0

  • Online ISBN: 978-1-0716-0231-7

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