小菜蛾血淋巴蛋白质双向电泳技术体系的建立及优化
Establishment and optimization of twodimensional electrophoresis system for proteome analysis of haemolymph in Plutella xylostella
蔡来来**，李灵顺，邹琦毛，钟鸣，王世贵***
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作者单位：杭州市动物进化与适应重点实验室 杭州师范大学杭州310036
中文关键词：双向电泳，蛋白质组学，小菜蛾
英文关键词：twodimensional electrophoresis, proteomics, Plutella xylostella
中文摘要： 采用不同方法对小菜蛾Plutella xylostella(L.)血淋巴进行双向电泳研究，建立了一套适用于小菜蛾血淋巴蛋白质组分析的双向电泳体系。从样品处理方案、等电聚焦时间、染色方法等因素对小菜蛾双向电泳结果的影响进行了分析和优化。结果表明，TCA/丙酮沉淀法提取蛋白损失少，图谱均匀清晰，分辨率和重复性更高。不同长度胶条的最佳上样量不同，胶条长度为7、17、24 cm时，最佳上样量依次为20~50 μg、50~300 μg、100~500 μg，而聚焦时间则分别以24 000、50 000、65 000 vhr为宜。第二向聚丙烯酰胺凝胶的浓度以12%为宜，电泳后蛋白点呈均匀分布。银染的灵敏度明显高于考马斯亮蓝染色，可以检测出更多的蛋白点，但考马斯亮蓝染色在后续蛋白点质谱鉴定中具有优势。
英文摘要：twodimensional electrophoresis system suitable for hemolymph proteome analysis in the diamondback moth, Plutella xylostella(L.), was developed. The effects of different experimental conditions; such as sample processing program, IEF time, staining methods, etc, on the results of twodimensional electrophoresis were analyzed and optimized. The results show that the TCA/acetone precipitation method had less protein loss, a clearer pattern, higher resolution and greater repeatability. Different lengths of gel strip needed different optimum amounts of sample protein and time; for strips 7, 17 and 24 cm in length the optimum amount of sample protein was 20-50 μg, 50-300 μg and 100-500 μg, respectively, and the appropriate focused time was 24 000 vhr, 50 000 vhr and 65 000 vhr, respectively. Strips were subjected to twodimensional electrophoresis after transfer onto 12% SDSpolyacrylamide gel in order to attain evenly distributed protein spots. Silver staining was significantly more sensitive than Coomassie brilliant blue, resulting in more protein spots being detected, however, Coomassie blue has the advantage that protein spots can be identified by mass spectrometry.