Nandakumar M P, Shen Jie, Raman Babu, Marten Mark R
Department of Chemical & Biochemical Engineering, University of Maryland, Baltimore County (UMBC), 1000 Hilltop Circle, Baltimore, Maryland 21250, USA.
J Proteome Res. 2003 Jan-Feb;2(1):89-93. doi: 10.1021/pr025541x.
In preparing intracellular microbial samples for one- or two-dimensional electrophoresis, trichloroacetic acid (TCA) precipitation is frequently used to remove interfering compounds. Solubilization of TCA precipitate typically requires the addition of a number of chaotropes or detergents, in a multistep process, that requires hours to carry out. In this study, a simple, rapid, one-step method to solubilize TCA precipitated proteins is presented. Precipitated proteins are pretreated with 0.2 M NaOH for less than 5 min, followed by addition of standard sample solubilization buffer (SSSB). When compared to solubilization with SSSB alone, NaOH pretreatment of TCA-precipitated intracellular protein from Aspergillus oryzae and Escherichia coli shows an approximate 5-fold increase in soluble protein. In addition, two-dimensional gel electrophoresis on resolubilized proteins shows an equivalent number of proteins in samples with and without NaOH pretreatment.
在制备用于一维或二维电泳的细胞内微生物样品时，经常使用三氯乙酸（TCA）沉淀法去除干扰化合物。TCA沉淀的溶解通常需要在多步过程中添加多种离液剂或去污剂，这一过程需要数小时才能完成。在本研究中，提出了一种简单、快速的一步法来溶解TCA沉淀的蛋白质。沉淀的蛋白质先用0.2 M NaOH预处理不到5分钟，然后加入标准样品溶解缓冲液（SSSB）。与仅用SSSB溶解相比，对米曲霉和大肠杆菌的TCA沉淀细胞内蛋白质进行NaOH预处理后，可溶性蛋白质增加了约5倍。此外，对重新溶解的蛋白质进行二维凝胶电泳显示，经过和未经过NaOH预处理的样品中的蛋白质数量相当。
