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-For efficient solubilization of hydrophobic membrane proteins-
The recent growing interest in analysis and identification of the total protein complement of a genome (proteomics) has prompted efforts in improving the existing techniques in two-dimensional gel electrophoresis (2-DGE). The invention of immobilized pH gradients (IPGs)1 to eliminate cathodic drift and the introduction of thiourea2 as a powerful chaotrope are two such examples. However, solubilization of hydrophobic, notably membrane-type, proteins remains a great challenge in 2-DGE. CHAPS is a sulfobetaine-type zwitterionic detergent which has been employed in combination with nonionic detergents (e.g. TRITON® X-100, NP-40) in 2-DGE to minimize the loss of membrane proteins due to hydrophobic interactions between the proteins (which can cause problems in protein extraction), and between the proteins and the IPG matrix (which can cause problems in the recovery of proteins in 2-DGE). Chevallet et al.3 have identified several new sulfobetaine-type zwitterionic detergents, among them ASB-14, ASB-16, and ASB-C8Ř, that show improved efficiency in protein solubilization in 2-DGE. Similar to CHAPS, these newly discovered detergents contain a polarized sulfobetaine head group, followed by a three-carbon linkage between the quaternary ammonium and the amido nitrogen. What makes them different from CHAPS is that they contain mainly linear hydrocarbon tails that are composed of 13 to 16 carbons. This allows them increased urea compatibility and, in some instances, improved membrane protein recovery in 2-DGE when compared with CHAPS. Henningsen et al.4 have shown that ASB-C8Ř was better than CHAPS at solubilizing an ion channel and a G-protein-coupled receptor. Using red blood cell ghosts as a model, Tastet et al.5 have shown that detergents such as ASB-14, ASB-16 and ASB-C8Ř give increased protein solubilization efficiency and, in general, better pattern and quality in 2-DGE than detergents with carboxybetaine hydrophilic heads or longer hydrophobic tails. |
