大菱鲆表皮蛋白质组双向电泳技术的建立及优化

Establishment and optimization of two-dimensional gel electrophoresis for epidermal proteome of turbot Scophthalmus maximus

通过对表皮蛋白质提取方法、不同裂解液配方、等点聚焦程序和上样量等条件的对比试验,建立及优化了大菱鲆表皮组织蛋白质的双向电泳体系。试验采用改良的TCA/丙酮和超声波提取相结合的方法提取大菱鲆表皮组织的全蛋白,裂解液配方根据Bio-Rad公司推荐的进行调整,使用17cmpH4~7的IPG胶条被动水化上样,上样量为250μg。等点聚焦程序采用30V线性6h、250V线性2.5h、500V线性1.5h、1000V快速2h、4000V线性3h、8000V快速3h、8000V线性1h、500V24h,凝胶银染后用I mage Master2D Platinum7.0软件进行分析,最终得到较满意的双向电泳图谱,具有较高的分辨率和重复性,为大菱鲆蛋白质组学的进一步研究奠定了基础。

The two-dimensional gel electrophoresis for the epidermal proteome of Scophthalmus maximus was established and optimized through comparative tests by using different extraction methods,isoelectric focusing programs and sample volumes.The total proteins of the turbot epidermis were extracted by the combination of improved trichloroacetic acid（TCA）/acetone and ultrasonic extractions.The lysis buffer based on the Bio-Rad recommendations was optimized.Protein of 250 μg was loaded on the 17cm pH 4-7 immobilized pH gradients（IPG）strips.The programs for the gel isoelectric focusing were 30V linear ramp,6h/250V linear ramp,2.5h/500V linear ramp,1.5h/1000V rapid,2h/4000V linear ramp,3h/8000V rapid,3h/8000V linear ramp,and 1h/500V 24h.The gel was stained with sliver nitrate,scanned and analyzed using the Image Master 2D Platinum 7.0 software.The two-dimensional gel electrophoresis analysis of the epidermal proteome of the turbot was successfully established with high quality map of separation under the optimization of the experimental conditions,which will be a valuable preparation for further epidermal proteomics research of S.maximus.