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Solubilization of Trichloroacetic Acid (TCA) Precipitated Microbial Proteins via NaOH for Two-Dimensional Electrophoresis

2002; American Chemical Society; Volume: 2; Issue: 1 Linguagem: Inglês

10.1021/pr025541x

1535-3907

M. P. Nandakumar, Jie Shen, Babu Raman, Mark R. Marten,

Protein purification and stability

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. Keywords: proteins • proteomics • Aspergillus oryzae • Escherichia coli