Isolation of Nucleoli

Sabine Hacot1, Yohann Coute2, Stéphane Belin1, Marie Alexandra Albaret1, Hichem C. Mertani1, Jean‐Charles Sanchez3, Manuel Rosa‐Calatrava1, Jean‐Jacques Diaz1

1 Université de Lyon, Lyon, France, 2 CEA, DSV, iRTSV, Laboratoire d'Etude de la Dynamique des Protéomes, Grenoble, France and Université Joseph Fourier, Grenoble, France, 3 Biomedical Proteomics Research Group, Département de Biologie Structurale et Bioinformatique, Centre Médical Universitaire, Geneva, Switzerland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.36
DOI:  10.1002/0471143030.cb0336s47
Online Posting Date:  June, 2010
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Abstract

Nucleoli are now recognized as multi‐functional nuclear domains involved in several fundamental cell processes such as ribosome biogenesis, regulation of the assembly of non‐ribosomal ribonucleoprotein complexes, tRNA maturation, sequestration of protein, viral infection, and cellular ageing. Extensive proteomic analyses of these nucleolar domains after their purification have contributed to the description of their multiple biological functions. Because nucleoli are the largest and densest nuclear structures, they are easily amenable to purification from nuclei of cultured animal cells using the protocol described in this unit. Curr. Protoc. Cell Biol. 47:3.36.1‐3.36.10. © 2010 by John Wiley & Sons, Inc.

Keywords: nuclear domain; nucleoli; cell fractionation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Nucleoli from Adherent Cell Cultures
  • Support Protocol 1: One‐Dimensional SDS Gel Electrophoresis of Proteins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation of Nucleoli from Adherent Cell Cultures

  Materials
  • 15 × 106 cultured adherent cells plated on 100‐ or 150‐mm Petri dishes
  • Dulbecco's phosphate buffered saline for cell culture (DPBS; appendix 2A), ice cold
  • Nucleoli standard buffer (NSB; see recipe)
  • 10% (v/v) Nonidet P‐40 (NP‐40)
  • 250 mM sucrose/10 mM MgCl 2
  • 880 mM sucrose/5 mM MgCl 2
  • 340 mM sucrose/5 mM MgCl 2
  • 0.34 M sucrose buffer
  • Cell scrapers (Biologix Research Company)
  • 15‐ml polypropylene centrifuge tubes
  • Refrigerated centrifuge (Jouan, CR422)
  • Micropipets
  • Phase contrast microscope
  • 0.4‐mm clearance Dounce homogenizer (Wheaton type; Kimble/Kontes)
  • Sonicator (Vibracell 72434; Bioblock Scientific)
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Figures

Videos

Literature Cited

Literature Cited
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