优化灰木相思组培苗蛋白质组的提取与分离技术

Optimized Techniques of Proteome Extraction and Separation from the in vitro culture of Acacia implexa

针对木本植物灰木相思(Acacia implexa Benth.)蛋白质分离过程中含有大量色素和酚等干扰物质的现象,本实验优化了各步实验条件。结果表明,在蛋白提取液中加入10%PVP,同时提高离心力、延长离心时间能提高可溶性蛋白得率,第一向等电聚焦24cm长的固相pH梯度胶条的最适蛋白上样量为1000μg,第二向SDS聚丙烯酰胺凝胶最适浓度为12.5%。优化后的实验配置体系提高了灰木相思组培苗总蛋白双向电泳的分辨率和重复性,建立一套适用于灰木相思组培苗蛋白质组提取与分离的方法。

A 2-DE （two-dimensional electrophoresis） protocol for total proteins of Acacia implexa Benth. was described. The yield of water-soluble proteins was increased if 10% PVP was added in the protein extraction buffer, centrifugal force was increased and centrifugal time was prolonged. The optimum loading quantity of sample proteins was 1 000 μg in isoelectric focusing. The optimum concentration of SDS-polyacrylamide gel electrophoresis was 12.5%. The optimized protocol markedly minimized the interference from wood pigments and other non-protein substances.