Organelle Proteomics

Pierre Lescuyer1, Mireille Chevallet1, Sylvie Luche1, Thierry Rabilloud1

1 CEA Grenoble, Grenoble
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 24.2
DOI:  10.1002/0471140864.ps2402s44
Online Posting Date:  June, 2006
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Abstract

This unit describes strategies for studying the proteomes of organelles, which is one example of targeted proteomics. It relies heavily on previously published units dealing with organelle preparation, protein solubilization, and proteomics techniques. A specific commentary for organelle proteomics is provided. Specific protocols for the isolation of nuclei from various sources (cell cultures, tissues) are also provided.

Keywords: proteomics; organelle; nuclei; mitochondria

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Workflow for Organelle Proteomics Analysis
  • Basic Protocol 2: Direct Isolation of Nuclei from Cultured Cells
  • Alternate Protocol 1: Direct Isolation of Nuclei from Cells with Walls or Tissues Using Mechanical Lysis
  • Alternate Protocol 2: Simultaneous Purification of Nuclei and Other Organelles for Proteomics Studies
  • Support Protocol 1: Evaluation of Nuclei Purity by Microscopic Examination
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Workflow for Organelle Proteomics Analysis

  Materials
  • Cells growing in tissue culture
  • Cell washing solution (see recipe), ice cold
  • Cell lysis solution (see recipe), ice cold
  • Nuclei wash solution (see recipe), ice cold
  • Nuclei storage solution (see recipe), ice cold
  • Liquid nitrogen
  • Refrigerated centrifuge

Basic Protocol 2: Direct Isolation of Nuclei from Cultured Cells

  • Tissue sample or cells with walls
  • Dounce glass‐glass homogenizer or blade‐type homogenenizer (e.g., Waring blender) for samples resistant to lysis

Alternate Protocol 1: Direct Isolation of Nuclei from Cells with Walls or Tissues Using Mechanical Lysis

  • Starting material: crude nuclei pellet provided by a detergent‐free cell lysis procedure (unit 4.3)
  • Cheesecloth

Alternate Protocol 2: Simultaneous Purification of Nuclei and Other Organelles for Proteomics Studies

  Materials
  • Microscopy stain solution (see recipe)
  • Nuclei suspension (see protocol 2 or Alternate Protocols protocol 31 or protocol 42)
  • Microscope slide and coverslip
  • Light microscope with 200× magnification
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Figures

Videos

Literature Cited

Literature Cited
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