海马组织蛋白质双向电泳条件实验研究

Optimization experimental condition of two dimensional gel electrophoresis technique for hippocampus

目的优化大鼠海马组织蛋白质双向凝胶电泳分离(2-DE)的实验条件。方法分离并比较不同大鼠海马组织总蛋白的制备方法,pH 3～10非线性胶条(NL)、pH 4～7的2种不同pH梯度预制胶条,300、700μg不同质量蛋白上样量,保存2个月及保存1年的标本对2-DE效果的影响。结果本研究采用裂解液及样本制备方法所得蛋白适于2-DE,海马组织总蛋白等电点(PI)主要在4～8;与300μg相比,700μg蛋白上样量对于信噪比控制效果更理想;新鲜标本中影响聚焦的物质较少,凝胶横纹少。结论新鲜海马组织匀浆经多次低温重复离心制备的蛋白,采用pH 4～7预制胶条(24 cm)及700μg上样量获得的2-DE图谱更理想。

Objective To optimize experimental condition of two dimensional gel electrophoresis （2-DE） technique for hippocampus. Methods Total protein samples were prepared from the isolated rat hippocampus and then determined by Bradford and separated by 2-DE. Comparisons were made on the effects of different sample preparations, including the pre-pH gradient strips （pH 3-10 NL, pH 4-7 ） , 300 p,g and 700 ug protein sample volumes and the storage period of 2 months and 1 year. Results In present study, protein preparation of hippocampus from lysis buffer formulations and multiple high-speed cryogenic cen- trifuge were suitable for 2-DE. Isoelectric point （PI） of hippocampus protein mainly ranged from pH 4 to 8. Compared with that of 300 p,g,700 ug protein sample volume was more ideal to control the signal to noise ratio （SNR）. Fresh samples less affected 2-DE map with fewer gel stripes. Conclusion Ideal 2-DE map can be harvested with the hippocampus proteomics technique of using fresh sample, pH 4-7 gradient strips and 700 ug protein sample volume.