橡胶树胶乳C-乳清双向电泳样品制备技术的建立及优化

Optimization of Protein Preparation Suitable for 2-DE of Hevea brasiliensis C-serum

在TCA/丙酮沉淀法的基础上,用乙醇和乙醚洗涤(称双乙法)沉淀获得橡胶树胶乳C-乳清蛋白样品,并在样品溶解时通过超声助溶进一步提高样品的溶解度。结果显示,利用改良后的样品制备方法(TCA/丙酮+双乙法+超声处理)所获得的C-乳清蛋白样品的溶解性大幅增加,蛋白得率高达8.47 mg/mL C-乳清,为传统TCA/丙酮法的1.6倍;同时降低了盐离子浓度,等电聚焦时升压顺利、聚焦完全,双向电泳的图谱质量明显改善,在银染2D胶上可获得1 206个蛋白点,为传统TCA/丙酮法的2.7倍。本文对胶乳C-乳清蛋白样品制备方法进行深入优化,获得了重现性较好的高质量双向电泳蛋白图谱,促进了橡胶树蛋白质组学研究。

The C-serum is a major component of the latex and a key material for proteinomics of Hevea brasiliensis. Using the 2-D electrophoresis, there are some disadvantages with protein extraction from C-serum, such as unsatisfied solubility, higher ions concentrations and too many impurities. In this paper, the alcohol-ether method and sonication were used to further remove interfering substances and solubilized protein based on the traditional method of TCA/acetone. Comparing the traditional method of extraction, the results showed that the new method improved the solubility, ions concentration and purity of protein samples from C-serum. The optimized methods for extraction of protein will be extremely useful for the proteomic research of C-serum form H. brasiliensis latex.