Sample preparation method for poorly soluble proteins prior to two-dimensional electrophoresis

• Published in: SEIBUTSU BUTSURI KAGAKU Vol. 48; no. 2; pp. 71 - 73
• Main Authors: Kameyama, Kazuhisa, Toda, Toshifusa
• Format: Journal Article
• Language: English
• Published: Japanese Electrophoresis Society 2004
• Subjects: poorly soluble proteins; sample preparation; SDS soluble proteins; two-dimensional electrophoresis; ultra filtration
• ISSN: 0031-9082; 1349-9785
• DOI: 10.2198/sbk.48.71

Protein sample for two-dimensional electrophoresis (2-DE) is conventionally extracted in a buffer containing urea and non-ionic detergent. However, poorly soluble proteins are hardly introduced to the first-dimensional isoelectric focusing (IEF) under the conventional denaturing condition. Moreover, some kinds of tissues and organs contain extraordinary amount of proteases and phosphatases, which reduce the reproducibility in two-dimensional protein profiling. Various kinds of detergents have been tested to solve the problem, and consequently sodium dodecyl sulfate (SDS) is realized as the best detergent because of its high solubilizing capacity. However, sample solution containing a high concentration of SDS can not be directly applicable to 2-DE because the anionic detergent disturbs the formation of stable pH gradient in the first-dimensional IEF. Therefore, it has been considered that buffer exchange is necessary to reduce SDS concentration in the sample solutions. And then, acetone and trichloroacetic acid (TCA) have been adopted for precipitation of proteins in SDS-containing samples. However, some of proteins precipitated in acetone or TCA are hardly re-solubilized in a 2-DE sample buffer. In this paper, ultra-filtration (UF) membrane was used for the buffer exchange without protein insolubilization. Consequently, proteins of high molecular weight were successfully detected on 2-DE gels when proteins were extracted in the SDS-containing buffer and treated with UF membrane to reduce the concentration of SDS prior to IEF. These results indicate that the protein extraction in SDS-containing buffer and subsequent buffer exchange using UF membrane are useful for 2-DE of poorly soluble proteins.