山黧豆叶片蛋白质双向电泳技术的建立

Establishment of Two-dimension Electrophoresis of Leaf Proteins of Lathyrus sativus

以山黧豆叶片为材料,比较分析了蛋白质的不同提取方法,在此基础上着重于样品制备.对IPG胶条的选择,第一向等电聚焦和第二向SDS-聚丙烯酰胺凝胶电泳的电泳程序及参数、染色方法等相关技术进行了比较和条件优化.结果显示:采用TCA-丙酮沉淀法提取蛋白质,裂解液中加入Tris-base作为蛋白酶抑制剂,等电聚焦电泳时延长低电压的电泳时间(30 V1、2 h,500 V1、h,1 000 V2、h)以促进盐离子泳出的方法对山黧豆叶片蛋白质进行双向电泳,并用考马斯亮蓝和银染复合染色法进行凝胶染色,能够获得蛋白点清晰的双向电泳图谱,说明用优化后的方法建立起的山黧豆叶片蛋白质双向电泳技术,蛋白质样品制备质量好,电泳分辨率高,完全适合于进一步的蛋白质组学研究.

Different protein extraction methods were comparatively examined with Lathyrus sativus leaves as the experimental material and on the basis of this the sample preparation was studied. The choices of IPG strips and the protocols,parameters and staining methods of the first phase IEF and the second-phase SDS-PAGE were compared and their conditions were optimized. The results showed that when TCA-acet precipitation was adopted to extract protein,Tris-base was added to the lysis solution as the protease inhibitor, when isoelectric focusing electrophoresis was adopted, the low voltage was prolonged（30 V, 12 h：500 V, 1 h; 1 000 V,2 h）to help the salt ion electrophoresis and thus conduct the two-dimension electrophorsis of leaf protein of L. sativus,and when Coomassie brilliant blue and compound silver staining were adopted to stain the gel, the two dimension electrophoretograms with high reproduction and resolution power of leaf protein of L. sativus were obtained, wihich indicated that the optimized two-dimension electrophoresis of leaf protein of L. sativus was characterized with quality sample preparation and high electrophoresis resolution thus being completely suited to further proteomics research.