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Proper solubilization after precipitation and keeping the purified proteins in solution during the whole separation process are very critical to achieve accurate and high resolution patterns in two-dimensional gel electrophoresis (2-DE). Chaotropes and detergents are embedded in the sample and rehydration buffers in order to prevent hydrophobic interactions between the hydrophobic protein domains and avoid loss of proteins due to aggregation and precipitation. Unfortunately, detergents used for IEF must bear no net electrical charge and only week nonionic and zwitterionic detergents may be used in this process. Because of the low solubility of proteins at or very close to their isoelectric point, it seems that choice of chaotropes and detergents can dramatically affect on2D separations, especially in the case of very hydrophobic proteomes. Considering the physico-chemical heterogeneity of tear film protein content, it is deemed that solubilization can play an important role in 2D tear proteome analysis. So herein, we investigated the effect of some various detergents and chaotropes on the solubility of tear proteome during the sample preparation and IEF process. The results illustrated a very poor performance of non-ionic detergents (Triton­X-100 and Tween­80). Zwitterionic detergents (CHAPS and SB­3-10) had a better solubilization power and provided more reliable 2D maps. Last of all a great improvement in spot number and 2D resolution is achieved using a combination of urea/Thiourea in rehydration buffer and application of SDS in the sample buffer with a modified protocol, which ensures complete removal of anionic detergent during the first step of IEF and its replacement with the zwitterionic CHAPS.  

Keywords: Tear proteome, IEF, 2-DE, Solubilization, Detergent, Chaotrope

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