蔗糖梯度离心技术方法在提取内质网的应用  

Method of Extracting Endoplasmic Reticulum by Sucrose Gradients Centrifugation

作　　者：付政祺[1,2] 杨莹[2] 田青[2] 

机构地区：[1]江汉大学医学院病理学与病理生理学教研室,湖北省武汉市430056 [2]华中科技大学同济医学院基础医学院病理生理学系

出　　处：《医学理论与实践》2011年第17期2022-2024,共3页The Journal of Medical Theory and Practice

摘　　要：目的:通过蔗糖梯度离心方法提取内质网,探讨在内质网提取过程中质量控制的关键要素。方法:本文利用蔗糖建立连续的密度梯度,通过超速离心提取HEK293/wt细胞中的内质网,并利用Western blot技术、内质网特异性抗体Bip/GRP78对内质网所处位置进行检测,确定内质网所在层面,成功提取高浓度、高质量的内质网。结果:通过对HEK293/wt细胞的破膜方法和离心时间进行调整,经Western blot技术检测,内质网位于蔗糖梯度的第10、11、12层。结论:选择合适的破膜方法和离心时间是成功提取高浓度、高质量内质网的关键。Objective：To explore the method and key elements of separating and enriching endoplasmic reticulum（ER） by sucrose gradients centrifugation.Methods：HEK293/wt cells were homogenized and fractionated on an equilibrium flotation sucrose gradient by centrifugation.Proteins from the lower 8 fractions（total 12 fractions）were precipitated and separated by 10% SDS/PAGE,followed by immunoblotting using antibodies against Bip/GRP78（specific antibody of ER）.Results：ER was found mainly in fractions of 10,11,and 12,after adjustments of methods of homogenization and centrifugation times.Conclusion：Appropriate methods of homogenization and time of centrifugation are the key to separate and enrich ER.

关 键 词：内质网提取 梯度离心 

分 类 号：G634.91[文化科学—教育学]