Preparation of envelope membrane fractions from Arabidopsis chloroplasts for proteomic analysis and other studies

Methods Mol Biol. 2011:775:189-206. doi: 10.1007/978-1-61779-237-3_10.

Abstract

Plastids are semiautonomous organelles restricted to plants and protists. These plastids are surrounded by a double membrane system, or envelope. These envelope membranes contain machineries to import nuclear-encoded proteins, and transporters for ions or metabolites, but are also essential for a range of plastid-specific metabolisms. Targeted semiquantitative proteomic investigations have revealed specific cross-contaminations by other cell or plastid compartments that may occur during chloroplast envelope purification. This article describes procedures developed to recover highly purified envelope fractions starting from Percoll-purified Arabidopsis chloroplasts, gives an overview of possible cross-contaminations, provides some tricks to limit these cross-contaminations, and lists immunological markers and methods that can be used to assess the purity of the envelope fractions.

MeSH terms

  • Arabidopsis / cytology*
  • Biomarkers / metabolism
  • Blotting, Western
  • Cell Fractionation / methods*
  • Chemical Fractionation
  • Chloroplasts / chemistry*
  • Chloroplasts / metabolism*
  • Electrophoresis, Polyacrylamide Gel
  • Intracellular Membranes / chemistry*
  • Intracellular Membranes / metabolism*
  • Mass Spectrometry
  • Plant Leaves / cytology
  • Povidone / chemistry
  • Proteomics / methods*
  • Silicon Dioxide / chemistry

Substances

  • Biomarkers
  • Percoll
  • Silicon Dioxide
  • Povidone