适合双向电泳的大白菜花蕾蛋白提取及浓度测定方法

The Best Method for Extracting and Concentration Detecting of Proteins Used for Two-dimensional Electrophoresis from Flowering Buds of Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

高质量提取大白菜花蕾蛋白是大白菜花器官蛋白质组学研究的关键步骤。本试验以大白菜AB01可育花蕾为材料,采用TCA丙酮沉淀法、Tris-HC1法、酚改良法、Trizol沉淀法、Tris-丙酮-酚法和尿素-硫脲提取法等六种方法分别提取花蕾全蛋白,通过Bradford法和双向电泳蛋白定量试剂盒测定蛋白浓度,经双向电泳分离后得到2-DE图谱,经分析、比较图谱蛋白点的情况,找到花蕾全蛋白的最佳提取方法和浓度测定方法。结果显示:TCA丙酮沉淀法得到的2-DE图谱,蛋白点分布均匀、清晰,是一种较为理想的花蕾蛋白提取方法;采用蛋白定量试剂盒测定蛋白浓度更加准确。

Extracting high-quality flower bud proteins is the prerequisite in studying of the floral-organ Proteome in Chinese cabbage. In this experiment, the flower buds of Chinese cabbage A/B line AB01 were used to extract the whole protein by using the following six methods （TCA acetone precipitation method, Tris-HC1 method, Phenol modified method, Trizol precipitation method, Tris-acetone-phenol method and Urea-thiourea extraction method）. The 2-DE maps were generated by the two-dimensional electrophoresis approach as well. The best methods for flower bud protein extraction and concentration determination were selected by analyzing and comparing the protein spots in 2-DE maps. The results showed that the protein spots in the 2-DE maps generated by TCA-acetone precipitation were clear and even distributed, therefore the TCA-acetone precipitation method was recommended as an ideal methods for extracting the flower bud protein, while the Protein Assay kit should be more accurate to detect the protein concentration based on comparing the maps generated by the two-dimensional electroDhoresis nrotein ouantitative kit and Bradford method.