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

한국식물학회지 2006년 49권 6호 p.413 ~ 420
 ( Cho Kyoung-Won ) - Chonnam National University Department of Applied Biotechnology

 ( Torres Nilka Lineth ) - University of Panama University Regional Center of Azuero
 ( Subramanyam Subhashree ) - Purdue University Department of Agronomy
 ( Deepak Saligrama A. ) - National Institute for Agro-Environmental Sciences
 ( Sardesai Nagesh ) - Purdue University Department of Biological Sciences
한옥수 ( Han Ok-Soo ) - Chonnam National University Department of Applied Biotechnology
 ( Williams Christie E. ) - Purdue University Department of Entomology
 ( Ishii Hideo ) - National Institute for Agro-Environmental Sciences
 ( Iwahashi Hitoshi ) - National Institute of Advanced Industrial Science and Technology Human Stress Signal Research Center
 ( Rakwal Randeep ) - National Institute of Advanced Industrial Science and Technology Human Stress Signal Research Center

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.

키워드

Arabidopsis; cucumber; lysis buffer; maize; rice; wheat
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