Protein extraction/solubilization protocol for monocot and dicot plant gel-based proteomics |
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Authors: | Kyoungwon Cho Nilka Lineth Torres Subhashree Subramanyam Saligrama A Deepak Nagesh Sardesai Oksoo Han Christie E Williams Hideo Ishii Hitoshi Iwahashi Randeep Rakwal |
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Institution: | (1) Department of Agronomy, Purdue University, West Lafayette, IN 47907, USA;(2) Department of Entomology, Purdue University, West Lafayette, IN 47907, USA;(3) Crop Production and Pest Control Research Unit USDA-ARS-MWA and Department of Entomology, Purdue University, West Lafayette, IN 47907, USA; |
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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. |
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Keywords: | Arabidopsis cucumber lysis buffer maize rice wheat |
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