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The Mitochondria of Cultured Mammalian Cells

II: Expression and Visualization of Exogenous Proteins in Fixed and Live Cells
  • Steffi Goffart
  • Peter Martinsson
  • Florence Malka
  • Manuel Rojo
  • Johannes N. Spelbrink
Part of the Methods in Molecular Biology™ book series (MIMB, volume 372)

Abstract

Mitochondria are almost ubiquitous organelles in Eukaryota. They are highly dynamic and often complex structures in the cell. The mammalian mitochondrial proteome is predicted to comprise as many as 2000–2500 different proteins. Determination of the subcellular localization of any newly identified protein is one of the first steps toward unraveling its biological function. For most mitochondrial proteins, this can now be done relatively easily by cloning a complementary deoxyribonucleic acid of interest in frame with an additional sequence for a fluorescent or nonfluorescent protein tag. Transfection and subsequent visualization, either by direct fluorescence microscopy or by indirect immunofluorescence microscopy, will give the first clue to mitochondrial localization. In combination with a fluorescent “marker” dye, the mitochondrial localization can be confirmed. This chapter describes some of the methods used in determining mitochondrial protein localization, which can also be used to study dynamics of mitochondria or individual mitochondrial proteins or protein complexes.

Key Words

DsRed fluorescent microscopy GFP mammalian cell culture MitoTracker PicoGreen transfection 

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Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Steffi Goffart
    • 1
  • Peter Martinsson
    • 1
  • Florence Malka
    • 2
  • Manuel Rojo
    • 2
  • Johannes N. Spelbrink
    • 1
  1. 1.FinMIT Centre of ExcellenceInstitute of Medical Technology and Tampere University Hospital, University of TampereTampereFinland
  2. 2.INSERM U582 -Institut de MyologieUniversité Pierre et Marie Curie, IFR14, Groupe Hospitalier Pitié-SalpêtrièreParis CedexFrance

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