Optimization of two-dimensional gel electrophoresis for proteome analysis of suspension-cultured ginseng cells

Abstract

Two-dimensional gel electrophoresis (2-DE) is the most widely used protocol to study protein expression and function in plants and plant diseases. In this study, optimized 2-DE sample preparation methodologies were established for suspension-cultured ginseng cells. Trichloroacetic acid–acetone (TCA–acetone), urea/thiourea and phenol extraction methods were evaluated for proteomic analysis of suspension cultures of ginseng. The solubilization buffer, sample volumes and concentration of polyacrylamide gels were also optimized in the 2-DE system. The phenol extraction method was proved to be the best method, which provided a greater spot resolution, a higher protein yield and a minimal streaking on 2-DE gels for suspension-cultured ginseng cells. A solubilization buffer containing 9 M urea, 2 M thiourea, 2% w/v CHAPS, 50 mM DTT, 1% TBP and 2% v/v IPG buffer at pH 3–10, yielded the best protein solubilization. High quality 2-DE protein expression profiles were obtained under the conditions of 0.8 mg sample mass loaded, 17 cm IPG strips (linear pH gradient 5–8), and 12% polyacrylamide gels. The optimized protocol is useful for comparative proteome analysis of suspension-cultured ginseng cells by 2-DE.