Advertisement

Native DIGE of Fluorescent Plant Protein Complexes

  • Veronika Reisinger
  • Lutz Andreas Eichacker
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 854)

Abstract

CyDye labeling and DIGE have not only been proven to work for soluble proteins but also at the level of whole membrane protein complexes. After complex solubilization and CyDye labeling, proteins can be separated by native PAGE which is often combined with SDS PAGE in a subsequent step. By this combination, sizes of complexes as well as their subunit composition can be compared after mixing samples from different physiological states. Plants interact specifically with light via protein-bound pigments. This can be used in combination with CyDye technology to extend the “classical” approach in plant research. As an example, chlorophyll can be excited for fluorescent scanning at the Cy5 excitation wavelength. This property can be used to identify pigment-binding plant complexes and complex subunits isolated from plastid membranes. In this protocol, we present a combination of the conventional CyDye labeling technique with 2D native/SDS PAGE and parallel scanning for CyDyes and fluorescence from endogenous bound chlorophyll for identification of pigment-binding complexes and complex subunits.

Key words

Native PAGE DIGE Membrane protein complexes Chlorophyll fluorescence Chlorophyll-binding proteins Plant Membrane Protein complexes Chlorophyll CyDye 

References

  1. 1.
    Marouga R, David S, Hawkins E (2005) The development of the DIGE system: 2D fluorescence difference gel analysis technology. Analytical and Bioanalytical Chemistry 382: 669–678.PubMedCrossRefGoogle Scholar
  2. 2.
    Heinemeyer J, Scheibe B, Schmitz UK, Braun HP (2009) Blue native DIGE as a tool for comparative analyses of protein complexes. J Proteomics 72: 539–544.PubMedCrossRefGoogle Scholar
  3. 3.
    Reisinger V, Eichacker LA (2007) How to analyze protein complexes by 2D blue native SDS-PAGE. Proteomics 7 Suppl 1: 6–16.PubMedCrossRefGoogle Scholar
  4. 4.
    Gillardon F, Rist W, Kussmaul L, Vogel J, Berg M, Danzer K, Kraut N, Hengerer B (2007) Proteomic and functional alterations in brain mitochondria from Tg2576 mice occur before amyloid plaque deposition. Proteomics 7: 605–616.PubMedCrossRefGoogle Scholar
  5. 5.
    Reisinger V, Hertle AP, Plöscher M, Eichacker LA (2008) Cytochrome b6f is a dimeric protochlorophyll a binding complex in etioplasts. FEBS Journal 275: 1018–1024.PubMedCrossRefGoogle Scholar
  6. 6.
    Rabilloud T (2009) Membrane proteins and proteomics: Love is possible, but so difficult. Electrophoresis 30: 174–80.CrossRefGoogle Scholar
  7. 7.
    Dreyfuss BW, Thornber JP (1994) Organization of the Light-Harvesting Complex of Photosystem I and Its Assembly during Plastid Development. Plant Physiol. 106: 841–848.PubMedGoogle Scholar
  8. 8.
    Wittig I, Braun HP, Schagger H (2006) Blue native PAGE. Nat Protoc 1: 418–428.PubMedCrossRefGoogle Scholar
  9. 9.
    Wittig I, Karas M, Schagger H (2007) High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes. Mol Cell Proteomics 6: 1215–1225.PubMedCrossRefGoogle Scholar
  10. 10.
    Wittig I, Schagger H (2005) Advantages and limitations of clear-native PAGE. Proteomics 5: 4338–4346.PubMedCrossRefGoogle Scholar
  11. 11.
    Krause F (2006) Detection and analysis of protein-protein interactions in organellar and prokaryotic proteomes by native gel electrophoresis: (Membrane) protein complexes and supercomplexes. Electrophoresis 27: 2759–2781.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Center of Organelle Research (CORE)University of StavangerStavangerNorway

Personalised recommendations