适用于黄麻根部蛋白质组学分析的双向电泳技术

以黄麻品种'9511'幼苗为试验材料,研究其根部蛋白提取方法的得率及不同的蛋白样品溶解方法、电泳上样量和IPG胶条pH范围对双向电泳图谱的影响.结果表明:采用三氯乙酸(TCA)/丙酮沉淀法提取黄麻根部蛋白质,蛋白得率为80 mg/g;蛋白粉末溶解采用两次水化法,裂解液中含有7 mol/L尿素、2 mol/L硫脲、4% CHAPS、65 mmol/L DTT、0.2%载体两性电解质和1 mmol/L PMSF,能够较充分地溶解蛋白质,且制备的样品浓度能够满足双向电泳上样要求;上样量为400 μg时得到的图谱分辨率高、蛋白斑点分布均匀、清晰;等电聚焦(Isoelectrofocusing,IEF)采用pH 4～7、17 cm的IPG胶条时所得图谱质量最佳.研究表明,样品的制备及IEF有效除盐对获得理想的2-DE图谱非常关键;取材、染色等细节对2-DE的重复性影响很大.

Two-Dimensional Electrophoresis Protocol for Root Proteomics Analysis of Orchorus olitorius

Genotype of jute '9511' was employed to study the changes of 2-DE picture under different methods for harvest rate of extraction of root protein and for dissolution of protein sample,quantitative loading of samples and pH range of IPG strip.The results indicated that the TCA/acetone precipitation method was the best one for jute root protein extraction with the extracting rate of 80 mg/g.The optimum dissolution method for protein samples was twi-lysis one with the buffer containing 7 mol/L urea,2 mol/L thiourea,4% CHAPS,65 mmol/L DTT,0.2% bio-lyte,and 1 mmol/L PMSF,which could dissolve the protein sample completely and the resultant protein concentration could satisfy 2-DE's the quantitative loading of samples.When the protein loading for 2-DE was 400 μg and the IPG (pH 4～7,17 cm) used for isoelectrofocusing (IEF),a good protein pattern with protein spots distributing equably and syllabify could be obtained.The results showed that the preparation for sample and wiping off salt in IEF had a key function for harvesting good 2-DE pictures;details which had great influence for reproducibility of 2-DE picture were obtaining materials,staining and so on.