The Nucleus pp 77-92 | Cite as

High-Yield Isolation and Subcellular Proteomic Characterization of Nuclear and Subnuclear Structures from Trypanosomes

  • Jeffrey A. DeGrasse
  • Brian T. Chait
  • Mark C. Field
  • Michael P. Rout
Part of the Methods in Molecular Biology book series (MIMB, volume 463)

Abstract

The vast evolutionary distance between the Opisthokonta (animals and yeast) and the excavata (a major group of protists, including Giardia and Trypanosoma) presents a significant challenge to in silico functional genomics and ortholog identification. Subcellular proteomic identification of the constituents of highly enriched organelles can alleviate this problem by both providing localization evidence and yielding a manageably sized proteome for detailed in silico functional assignment. We describe a method for the high-yield isolation of nuclei from the kinetoplastid Trypanosoma brucei. We also describe the subsequent purification of subnuclear compartments, including the nuclear envelope and nucleolus. Finally, using several proteomic strategies, we survey the proteome of a subcellular structure or organelle, using the nuclear pore complex as an example.

Keywords

Nuclear isolation Trypanosoma brucei Subcellular fractionation Proteomics Sucrose density gradient centrifugation Nuclear envelope Nucleolus Nuclear pore complex 

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Copyright information

© Humana Press 2008

Authors and Affiliations

  • Jeffrey A. DeGrasse
    • 1
  • Brian T. Chait
    • 1
  • Mark C. Field
    • 2
  • Michael P. Rout
    • 3
  1. 1.Laboratory of Mass Spectrometry and Gaseous Ion ChemistryThe Rockefeller UniversityNew YorkUSA
  2. 2.Department of PathologyUniversity of CambridgeCambridgeUK
  3. 3.Laboratory of Cellular and Structural BiologyThe Rockefeller UniversityNew YorkUSA

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