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

Cho, K. and Torres, N. L. and Subramanyam, S. and et al, . (2006) Protein extraction/solubilization protocol for monocot and dicot plant gel-based proteomics. Journal of Plant Biology, 49 (6). pp. 413-420.

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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.

Item Type: Article
Additional Information: This work was supported by institutional funds from the HSS.
Uncontrolled Keywords: Arabidopsis, cucumber, lysis buffer, maize, rice, wheat
Author Affiliation: Chonnam National University Department of Applied Biotechnology, Agricultural Plant Stress Research Center, and Biotechnology Research Institute 500-757 Kwangju Korea
Subjects: Crop Improvement
Divisions: General
Depositing User: Mr Siva Shankar
Date Deposited: 17 Sep 2012 08:26
Last Modified: 17 Sep 2012 08:28
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