摘要
为了探索并建立刺参的体腔液蛋白双向电泳体系,实验对刺参体腔液的蛋白质制备方法、上样量、IPG胶条的pH选择及等电聚焦程序进行了优化,并利用银染法进行染色。结果表明：改进的TCA-丙酮沉淀法增加了提取的蛋白量和纯度;采用8.5 cm,pH 4~6.5的IPG胶条,45μg蛋白上样量,等电聚焦先设250 v电压,7 mA电流,聚焦时间30 min后,再将电压升至1000 v,聚焦时间3 h,能有效提高双向电泳（2-DE）图谱中蛋白点的分离度和分辨率。本实验可用于筛选刺参差异表达蛋白以及后续的刺参蛋白质组学研究。
In order to use 2-DE analysis in the coelomic fluid proteins of Apostichopus japonicus. The protein extraction methods, sample volume, pH of IPG gel strips and isoelectric focusing programs were optimized to obtain best results. The results showed that the protein yield and purity were increased by improved TCA- acetone precipitation treatment. The resolution and reproducibility of 2-DE profiles were significantly improved by electing 8.5 cm （ pH 4-6.5） IPG gel strips, loading the sample 45 txg and seting 250 v, 7 mA isoelectric focusing for 30 min then 1000 v for 3 h. The result offered an important reference in screening differential expression protein and proteomics in Apostichopusjaponicus.
出处
《中国农学通报》
CSCD
2014年第14期39-45,共7页
Chinese Agricultural Science Bulletin
基金
国家自然科学基金项目"刺参盐度调控基因的筛选及功能验证"(41106128)
辽宁省高等学校青年学者成长支持计划"盐度胁迫下刺参生理特性及分子调控机制研究"(LJQ2012064)
国家"863"计划项目"高值海珍品良种培育"(2012AA10A412)
关键词
刺参
体腔液
双向电泳
Apostichopusjaponicus
coelomic fluid
protein two-dimensional electrophoresis （2-DE）
