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United States Department of Agriculture

Agricultural Research Service

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

Authors
item Cho, K - CHONNAM NATIONAL UNIV.
item Tores, N - HUMAN STRESS RES CTR
item Subramanyam, S - PURDUE UNIVERSITY
item Deepak, S - NATL INST KANNONDAI JAPAN
item Sardesai, N - PURDUE UNIVERSITY
item Han, O - CHONNAM NATL UNIV, KOREA
item Williams, Christie
item Ishii, H - NATL INST AGRO-ENVIRO
item Kubo, A - NATL INST ENVIRO STUDIES
item Iwahashi, H - NATL INST IND SCI & TECH

Submitted to: Springer Verlag
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 9, 2006
Publication Date: December 1, 2006
Citation: Cho, K., Tores, N.L., Subramanyam, S., Deepak, S.A., Sardesai, N., Han, O., Williams, C.E., Ishii, H., Kubo, A., Iwahashi, H. 2006. Protein extraction/solubilization protocol for monocot and dicot plant gel-based proteomics. Springer Verlag. 49:413-420.

Interpretive Summary: Purifying plant proteins for 2-dimensional gel analysis is difficult and often leads to partially degraded proteins. A new protein extraction protocol and a new gel technique were found to make a big difference in the quality of the data gleaned from 2-dimensional gels. More proteins were solubilized than with standard methods, resulting in higher spot number and resolution on gels. Indicator proteins were identified. This method is useful to other scientists because it works with a wide range of diverse plant species and solves a common problem in sample preparation.

Technical Abstract: Sample preparation in plant proteomics is tedious and requires modifications depending on the type of plant tissue. Here we present a standardized protein extraction protocol for use in both monocotyledonous (monocot) and dicotyledonous (dicot) plants, which significantly improves solubilization of total proteins. Using primarily leaf tissue and seed from the cereal crop genome model plant, rice, as an example, 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). The separation of leaf total proteins by two-dimensional gel electrophoresis (2-DE) revealed improved solubilization and increased spot numbers, visualized with Coomassie brilliant blue (CBB) staining, and better resolution over the use of LB-TT alone for protein extraction. The seed proteins could be extracted in LB-TT itself without need for TCAAEB, which resulted in a highly insoluble precipitate. Our CBB-stained 2-D gel protein profiles reveal the efficacy of this protein extraction protocol for total protein extraction/solubilization from the dicot genome model (Arabidopsis), a dicot disease model (cucumber), and two important monocot cereal crop models (maize and wheat). This is also a first report for generating a clean 2-D gel proteome profile for wheat crown and cucumber leaf tissues. Finally, as one of the goals of gel-based proteomics is modification and functional proteomics, we 1) coupled 2-DE with immunoblotting using a monoclonal anti-ascorbate peroxidase (APX) antibody, to identify cross-reacting and differentially expressed potential APX proteins, 2) show the suitability of a phosphoprotein detection dye (Pro-Q Diamond Phosphoprotein Gel Stain) to identify the phosphoproteins on 2-D gels, and 3) provide rice leaf and wheat crown LB-TT solubilized protein high-resolution large-format silver stained 2-D gel reference maps, as examples.

Last Modified: 8/27/2014
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