泥蚶肌肉组织蛋白质双向电泳体系的建立

Preliminary establishment of two-dimensional gel electrophoresis(2-DE) for muscle tissue of bloody clam Tegillarca granosa

本研究建立了泥蚶肌肉组织蛋白质的双向电泳体系．提取泥蚶肌肉组织全蛋白，进行了双向电泳分离的初步尝试，并对各个关键因素和环节进行了优化比较．优化的裂解液配方是8mol／dm。Urea，1．5mol／dm^3 Thioruea，4％ CHAPS，10mmol／dm^3 Tris，2mmol／dm^3 TBP，0．2％Bio-Lyte 3／10ampholyte，1mmol／dm^3 PMSF以及1mmol／dm^3 EDTA．使用pH值为4～7的IPG胶条进行被动水化上样，等电聚焦采用缓慢升压模式，电泳参数设置和染色方法等根据Bio—rad公司推荐的进行调整．最终获得了较满意的双向电泳图谱，具有较高的分辨率和重复性，成功建立了泥蚶肌肉组织蛋白质双向电泳分离体系．

The two-dimensional gel electrophiresis （2-DE） for proteins in muscle tissue of Tegillarca granosa Linnaeus was established and optimized. The proteins were extracted from fresh muscle tissue of T. granosa, and the lysis buffer containing 8mol/dm3 Urea, 1.5mol/dm^3 Thioruea,4% CHAPS, 10mmol/dm3Tris ,2mmol/dm3TBP ,0. 2% Bio-Lyte 3/10 ampholyte ,lmmol/dm3 PMSF and lmmol/dm3 EDTA. Two hundred micrograms of protein was loaded on 7cm IPG strip holder and run with isoelecrtic focusing electrophoresis as the first dimension and then vertical SDS-PAGE as the second dimension. Factors, such as sample handling, gel preparation, lysis solution, IPG gel rehydration,electrophoresis parameters, staining and so on were optimized. After all factors were optimized, we successfully established the 2-DE patterns of T. granosa with good separation features.