Abstract: The plant materials used in these experiments were the flourishing late tobacco leaves of Zhongyan 100, a drought-sensitive variety, and the seedling leaves of Xiaohuangjin 1025, a variety susceptible for the tobacco black shank pathogen. Total proteins were extracted by trichloroacetic acid/acetone precipitation method, respectively, and protein fractionated extraction combined with phenol extraction and acetone washing method with some minor modifications. A trichloroacetic acid/acetone precipitation method, used for protein extraction from Zhongyan 100 leaves, was optimized. The immobilized pH gradient strips applied to Zhongyan 100 total proteins was selected preliminarily. On the basis of this study, we compared the effects of different protein quantities, from Xiaohuangjin 1025 total proteins extracted by the second protein extraction method, on 2-DE profile. 2-DE experiment protocol suitable for tobacco leaves proteome study was established preliminarily.