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.