Purification of Ribosomes from Human Cell Lines

Stéphane Belin1, Sabine Hacot2, Lionel Daudignon2, Gabriel Therizols2, Stéphane Pourpe2, Hichem C. Mertani2, Manuel Rosa‐Calatrava2, Jean‐Jacques Diaz2

1 Boston Children Hospital/Harvard Medical School, Boston, Massachusetts, 2 Université de Lyon, Lyon, France
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.40
DOI:  10.1002/0471143030.cb0340s49
Online Posting Date:  December, 2010
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Abstract

Highly conserved during evolution, the ribosome is the central effector of protein synthesis. In mammalian cells, the ribosome is a macromolecular complex composed of four different ribosomal RNAs (rRNA) and about 80 ribosomal proteins. Requiring more than 200 factors, ribosome biogenesis is a highly complex process that takes place mainly within the nucleoli of eukaryotic cells. Crystallographic data suggest that the ribosome is a ribozyme, in which the rRNA catalyses the peptide bond formation and ensures quality control of the translation. Ribosomal proteins are involved in this molecular mechanism; nonetheless, their role is still not fully characterized. Recent studies suggest that ribosomes themselves and/or the mechanisms underlying their synthesis, processing, and assembly play a key role in the establishment and progression of several human pathologies. The protocol described here is simple, efficient, and robust, and allows one to purify high‐quality ribosomes from human cultured cell lines. Ribosomes purified with this protocol are adequate for most of the subsequent analyses of their RNA and protein content. Curr. Protoc. Cell Biol. 49:3.40.1‐3.40.11. © 2010 by John Wiley & Sons, Inc.

Keywords: ribosome; cell fractionation; translation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Purification of Ribosomes
  • Support Protocol 1: One‐Dimensional SDS Polyacrylamide Gel Electrophoresis (SDS‐PAGE)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Purification of Ribosomes

  Materials
  • Cultured adherent cells
  • Appropriate medium for cells to be cultured
  • Dulbecco's phosphate‐buffered saline (DPBS; appendix 2A)
  • Buffers A, B, and C (see reciperecipes)
  • 10% (v/v) Nonidet P‐40
  • 4 M KCl
  • Sucrose cushion (see recipe)
  • 12.5% SDS‐PAGE gel (Gallagher, )
  • 150‐mm diameter Petri dishes
  • 15‐ and 50‐ml conical polypropylene centrifuge tubes (e.g., BD Falcon)
  • Cell lifters (Biologix Research Company; http://biologixresearch.com/)
  • Refrigerated centrifuge (e.g., Jouan CR422) for cell harvesting
  • 3‐ml thick‐walled polycarbonate tubes (Beckman)
  • Refrigerated ultracentrifuge with fixed‐angle rotor (e.g., Beckman TL‐100.3) for ribosome harvesting
  • Spectrophotometer capable of measuring at 260 nm
  • Additional reagents and equipment for protein quantification (see protocol 2 and Simonin and Smith, ), SDS‐PAGE (see protocol 2 and Gallagher, ), and staining gels (see protocol 2 and Sasse and Gallagher, )
NOTE: Carry out all procedures at 4°C using prechilled solutions.NOTE: This protocol is optimized for dissociated cytoplasmic ribosomal subunits. To purify polysomes, 40S and 60S subunits, or monosome 80S, buffers have to be modified. See for more details.
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Figures

Videos

Literature Cited

Literature Cited
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