水牛精子蛋白质组双向电泳体系的建立和优化

建立和优化一种适合水牛精子蛋白质组学研究的双向电泳技术。以水牛精子为研究对象,比较两种不同配方的裂解液,以及不同上样量对其2-DE图谱质量的影响。结果显示,以7 mol/L尿素、2 mol/L硫脲、4%CHAPS、1%DTT、0.5%Cocktail of protease inhibitors为裂解液,24 cm胶条上样量200μg时,可获得较好的精子总蛋白质2-DE图谱。运用ImageMaster 2-Dplatinum分析软件检测出约500个蛋白质点,蛋白质大部分分布在等电点5-7之间,分子量范围约40-90 kD。

Establishment and Optimization of a Two-Dimensional Electrophoresis Technique for Proteome of Buffalo Sperm

Two dimensional gel electrophoresis is one of core technologies for proteomics studies and can be used for separating sperm proteins.However,the resolution of 2D maps is highly influenced by different experiment conditions.In order to establish and optimize a two-dimensional electrophoresis technique for buffalo spermatoza proteome analysis.The effects of two different lysis buffers lysing spermatoza and different loading volumes(150 μg,200 μg,250 μg)on the quanty of the 2-D maps were studied.The results showed that a clearly 2-DE map was obtained when the buffalo spermatoza were lysed by lysis buffer B(7 mol/L Urea,2 moL/L Thiourea,4%CHAPS,1% DTT and 0.5% Cocktail of protease inhibitors) and 200 μg loading volume were used in the 24 cm IPG strip(pH3-10).About 500 spots were detected by the Image Master 2-D platinum software,and the analysis result showed that isoelectric point and molecular weight of most proteins were localized in 5 to 7 kD and 40-90 kD.So a two dimensional electrophoresis workflow for buffalo spermatoza proteins separation has been established,which could laid a foundation for the further study on differential proteomics of buffalo spermatoza.