Separation of the Plant Golgi Apparatus and Endoplasmic Reticulum by Free-Flow Electrophoresis

  • Harriet T. Parsons
  • Susana M. González Fernández-Niño
  • Joshua L. Heazlewood
Part of the Methods in Molecular Biology book series (MIMB, volume 1072)


Free-flow electrophoresis (FFE) is a technique for separation of proteins, peptides, organelles, and cells. With zone electrophoresis (ZE-FFE), organelles are separated according to surface charge. The plant Golgi and endoplasmic reticulum (ER) are similar in density and are therefore separated with difficulty using standard techniques such as density centrifugation. Purification of the ER and Golgi apparatus permits a biochemical and proteomic characterization which can reveal the division of processes between these compartments. Here we describe complete separation between the ER and more negatively charged Golgi compartments using ZE-FFE. We also describe techniques for assigning proteins to partially separated ER and the less negatively charged Golgi compartments.

Key words

Free-flow electrophoresis Golgi apparatus Arabidopsis Endoplasmic reticulum Proteomics 



This work conducted by the Joint BioEnergy Institute was supported by the Office of Science, Office of Biological and Environmental Research, of the US Department of Energy under Contract No. DE-AC02-05CH11231.


  1. 1.
    Eubel H, Lee CP, Kuo J et al (2007) Free-flow electrophoresis for purification of plant mitochondria by surface charge. Plant J 52: 583–594PubMedCrossRefGoogle Scholar
  2. 2.
    Zischka H, Weber G, Weber PJA et al (2003) Improved proteome analysis of Saccharomyces cerevisiae mitochondria by free-flow electrophoresis. Proteomics 3:906–916PubMedCrossRefGoogle Scholar
  3. 3.
    Eubel H, Meyer EH, Taylor NL et al (2008) Novel proteins, putative membrane transporters, and an integrated metabolic network are revealed by quantitative proteomic analysis of Arabidopsis cell culture peroxisomes. Plant Physiol 148: 1809–1829PubMedCrossRefGoogle Scholar
  4. 4.
    Islinger M, Li KW, Loos M et al (2010) Peroxisomes from the heavy mitochondrial fraction: isolation by zonal free flow electrophoresis and quantitative mass spectrometrical characterization. J Proteome Res 9:113–124PubMedCrossRefGoogle Scholar
  5. 5.
    Parsons HT, Christiansen K, Knierim B et al (2012) Isolation and proteomic characterization of the Arabidopsis Golgi defines functional and novel targets involved in plant cell wall biosynthesis. Plant Physiol 159:12–26PubMedCrossRefGoogle Scholar
  6. 6.
    Islinger M, Eckerskorn C, Volkl A (2010) Free-flow electrophoresis in the proteomic era: a technique in flux. Electrophoresis 31: 1754–1763PubMedCrossRefGoogle Scholar
  7. 7.
    Mitsui T, Kimura S, Igaue I (1990) Isolation and characterization of Golgi membranes from suspension-cultured cells of rice (Oryza sativa L). Plant Cell Physiol 31:15–25Google Scholar
  8. 8.
    Dunkley TPJ, Hester S, Shadforth IP et al (2006) Mapping the Arabidopsis organelle proteome. Proc Natl Acad Sci USA 103: 6518–6523PubMedCrossRefGoogle Scholar
  9. 9.
    Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15: 473–497CrossRefGoogle Scholar
  10. 10.
    Heazlewood JL, Verboom RE, Tonti-Filippini J et al (2007) SUBA: the Arabidopsis subcellular database. Nucleic Acids Res 35:D213–D218PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Harriet T. Parsons
    • 1
  • Susana M. González Fernández-Niño
    • 1
  • Joshua L. Heazlewood
    • 1
  1. 1.Joint BioEnergy Institute and Physical Biosciences DivisionLawrence Berkeley National LaboratoryBerkeleyUSA

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