橡胶树C-乳清三种蛋白提取方法双向凝胶电泳分析

摘要：C-乳清是胶乳的主要组分，就双向凝胶电泳技术而言，目前仍无有效的蛋白提取方法，这严重制约了双向凝胶电泳技术在橡胶树蛋白组分析上的应用。本文选取3种方法提取C-乳清蛋白，通过对蛋白产量和提取蛋白的双向凝胶电泳图谱分析系统评价3种方法。就蛋白产量而言，三氯乙酸/丙酮沉淀法产量最高（0.86 mg/ml C-乳清），三氯乙酸沉淀法次之（0.72 mg/ml C-乳清），酚提取法最低（0. 66 mg/ml C-乳清）。双向凝胶电泳图谱分析表明：酚提取法可检测到447个蛋白点，图谱背景较暗且横纵向纹理多；三氯乙酸沉淀法可检测到821个蛋白点，图谱背景居中；三氯乙酸/丙酮沉淀法可检测到1052个蛋白点，图谱背景清晰且基本无纹理。综上所述，三氯乙酸/丙酮沉淀法最适合C-乳清蛋白提取。本文首次系统评价C-乳清蛋白提取方法，必将推动橡胶树C-乳清蛋白研究发展。

Evaluation of three protein extraction methods for Hevea brasiliensis C-serum suitable for 2-DE

Abstract: The C-serum is a major component of the latex, but there is no effective protein extraction method from the latex C-serum for 2-DE, which badly limits the utilization of the 2-DE on protein analysis of rubber tree. In this paper, three protein extraction methods were used to extract protein from the latex C-serum. Analyzing the protein yield and quality for the 2-DE, we systematically evaluated three protein extraction methods. As for protein yield, TCA/acetone method showed the highest protein yield (0.86 mg protein/ml C-serum), followed by E-TCA method (0.72 mg protein/ml C-serum) and phenol extraction method (0.66 mg protein/ml C-serum). On 2-DE gel from phenol extraction method, only 447 protein spots were detected with a dark background and considerable vertical and horizontal streaking. When the sample from E-TCA method was loaded, there were about 821 protein spots separated on the gel, and the gel showed less contamination. In contrast with phenol extraction method and E-TCA method, the TCA/acetone method indicated about 1052 protein spots with clear background and almost free of streaking on the 2-DE gel. In conclusion, the TCA/acetone method was more suitable for the latex C-serum protein extraction for 2-DE. To our knowledge, it is the first time that the protein extraction methods from the latex C-serum for 2-DE have systematically been reported, which is expected to accelerate the proteomic study of the latex C-serum.