摘要
目的:一种适用于双向电泳体系的松材线虫全蛋白提取方法的建立及其双向电泳体系的优化。方法:以松材线虫为实验材料,比较2种不同的蛋白提取方法,并对双向电泳中的IPG胶条长度、IPG胶条最适pH范围、上样量等3个方面的条件进行优化。结果:采用TCA-丙酮法提取的蛋白质浓度较高,达到2.18μg/μl。使用pH 5～8、24cm的IPG干胶条,上样量为120μg,经双向电泳分离可得到背景清晰、分辨率较高的2-DE图谱,能检测到2 000个左右清晰的蛋白点,含有相对丰富的蛋白信息量。结论:该实验所建立的松材线虫提取方法和优化体系可以为今后松材线虫蛋白质组学的研究奠定技术基础。
Objective：Extraction of total protein from Bursaphelenehus xylophilus and establishment of the optimized two-demensional eletrophoresism system.Method：With B.xylophilus as the study material,we compared two different protein extraction methods—trichloroacetic acid（TCA）/acetone and 2-D Clean Up Kit.The key factors of 2-DE electrophoresis,such asthe length of IPG strip,the pH gradient of IPG strip and sample loading were optimized.Result：Compared to 2D Clean Up Kit,total proteins acquired by TCA/acetone precipitation had a higher concentration（2.18μg/μl）.Extracting the total protein from B.xylophilus by TCA/acetone precipitation,separating the proteins with 24 cm pH 5~8 NL IPG strips,loading protein samples of 120 μg and staining the gels by silver staining after two-dimensional electrophoresis.The map of high quality with high resolution were obtained by this optimized 2-DE system of B.xylophilus,approximately 2 000 protein spots were distinguished on the 2-DE map.Conclusion：The developed method in this study can become an imporant tool for suduying Bursaphelenehus xylophilus proteomics in the future.
出处
《生物技术》
CAS
CSCD
北大核心
2011年第5期58-62,共5页
Biotechnology
基金
国家林业局公益项目(200704026)
国家自然科学基金项目(31000303)
江苏省自然科学基金项目(BK2010566)资助
关键词
松材线虫
蛋白质提取
双向电泳
Bursaphelenehus xylophilus
protein extraction
two-dimensional eletrophoresis
