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Journal of Plant Biology

, Volume 49, Issue 6, pp 413–420 | Cite as

Protein extraction/solubilization protocol for monocot and dicot plant gel-based proteomics

  • Kyoungwon Cho
  • Nilka Lineth Torres
  • Subhashree Subramanyam
  • Saligrama A Deepak
  • Nagesh Sardesai
  • Oksoo Han
  • Christie E. Williams
  • Hideo Ishii
  • Hitoshi Iwahashi
  • Randeep Rakwal
Article

Abstract

Sample preparation in plant proteomics is tedious, requiring modifications depending on the type of tissue involved. Here, we describe a protein extraction protocol for both monocotyledonous (monocot) and dicotyledonous (dicot) species, which significantly improves the solubilization of total proteins. For example, we used the primary leaf tissue and seeds from rice, a cereal crop and genome model system. Total protein was first precipitated with trichloroacetic acid/acetone extraction buffer (TCAAEB) and subsequently solubilized with a modified O’Farrell lysis buffer (LB) containing thiourea and tris (LB-TT). Separation of total leaf proteins by two-dimensional gel electrophoresis (2-DGE) revealed improved solubilization, as determined by an increased number of spots detected with Coomassie brilliant blue (CBB) staining. In addition, the resolution was better than when LB-TT was used alone for protein extraction. Seed proteins could be extracted in LB-TT itself without the need for TCAAEB, which resulted in a highly insoluble precipitate. Our CBB-stained 2-D gel protein profiles also demonstrated the efficacy of this protocol for total protein extraction/solubilization from the dicot genome model (Arabidopsis), a dicot disease model (cucumber), and two other important monocot cereal crop models (maize and wheat). Moreover, this is the first report on generating a 2-D gel proteome profile for wheat crown and cucumber leaf tissues. Finally, as examples of proteome reference maps, we obtained silver nitrate-stained, large-format 2-D gels for rice leaf and wheat crown LB-TT solubilized proteins.

Keywords

Arabidopsis cucumber lysis buffer maize rice wheat 

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Copyright information

© The Botanical Society of Korea 2006

Authors and Affiliations

  • Kyoungwon Cho
    • 1
  • Nilka Lineth Torres
    • 2
  • Subhashree Subramanyam
    • 3
  • Saligrama A Deepak
    • 4
  • Nagesh Sardesai
    • 5
  • Oksoo Han
    • 1
  • Christie E. Williams
    • 6
    • 7
  • Hideo Ishii
    • 4
  • Hitoshi Iwahashi
    • 8
  • Randeep Rakwal
    • 8
  1. 1.Department of Applied Biotechnology, Agricultural Plant Stress Research Center, and Biotechnology Research InstituteChonnam National UniversityKwangjuKorea
  2. 2.University Regional Center of AzueroUniversity of PanamaProvince of HerreraPanama
  3. 3.Department of AgronomyPurdue UniversityWest LafayetteUSA
  4. 4.National Institute for Agro-Environmental SciencesIbarakiJapan
  5. 5.Department of Biological SciencesPurdue UniversityWest LafayetteUSA
  6. 6.Department of EntomologyPurdue UniversityWest LafayetteUSA
  7. 7.USDA-ARS Crop Production and Pest Control Research UnitPurdue UniversityWest LafayetteUSA
  8. 8.Human Stress Signal Research CenterNational Institute of Advanced Industrial Science and Technology WESTIbarakiJapan

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