摘要
目的:建立适用于酸浆种子的SDS-PAGE和2-D PAGE蛋白样品提取方法。方法:比较直接提取法、TCA-丙酮法、Tris-丙酮法、饱和酚法、改良饱和酚法等7种提取方法所获酸浆种子总蛋白含量以及SDS-PAGE电泳的谱带数目、强度和电泳分辨率等方面的差异。结果:HEPES buffer直接提取法提取蛋白效率较高,杂质少,电泳谱带清晰,分辨率高;PVP buffer和Lysis buffer A直接提取法次之,但杂质较多;TCA-丙酮法、Tris-丙酮法、饱和酚法和改良的饱和酚法差别不大,蛋白纯度较高,但提取效率低,谱带数目少;此外,Lysis buffer A法、丙酮法和酚法易产生高丰度蛋白的干扰。结论:直接提取法中的HEPES buffer法最好,优于丙酮法、酚法和其它直接提取法。
Objective： To compare seven protein extraction protocols （PVP buffer, HEPES buffer, Lysis buffer A extraction and TCA/acetone, tris-base/acetone, phenol/ammonium acetate, acetone/ phenol/ammonium acetate precipitation） and establish a suitable one for total proteins extraction from Semen Seu Fructus Physalis. Methods： The protein yield, protein purity and SDS-PAGE patterns was determined and analyzed. Results： HEPES buffer extraction produce the purer sample; much more clear bands and highest resolution. PVP buffer and Lysis buffer A extraction had most impurities. TCA/ acetone, tris-base/acetone, phenol/ammonium acetate, acetone/phenol/ ammonium acetate precipitation methods had the purest sample, but protein yield and band numbers were lowest. Besides, the methods employing Lysis buffer was easy to produce high-abundance proteins and interfered in the electrophoresis resolution. Conclusion： HEPES buffer extraction was the best sample preparation protocol for SDS-PAGE and 2-D PAGE.
出处
《中国实验方剂学杂志》
CAS
北大核心
2010年第1期14-17,共4页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家科技基础条件平台项目(2005DKA21004)
