Towards the recovery of hydrophobic proteins on two-dimensional electrophoresis gels

An extensive proteomic approach relies on the possibility to visualize and analyze various types of proteins, including hydrophobic proteins which are rarely detectable on two-dimensional electrophoresis (2-DE) gels. In this study, two methods were employed for the purification of hydrophobic protei ns from Arabidopsis thaliana leaf plasma membrane (PM) model plants, prior to analysis on 2-DE immobilized pH gradient (IPG) gels. Solubilization effi ciency of two detergents, (3-[(3-cholomidopropyl)-1-propanesulfonic acid (C HAPS)]) and C8O, were tested for the recovery of hydrophobic proteins. An i mmunological approach was used to determine the efficiency of the above met hods. Fractionation of proteins by Triton X-114 combined with solubilizatio n with CHAPS resulted in the inability to detect hydrophobic proteins on 2- DE gels. The use of C8O for protein solubilization did not improve this res ult. On the contrary, after treatment of membranes with alkaline buffer, th e solubilization of PM proteins with detergent C8O permitted the recovery o f such proteins on 2-DE gels. The combination of membrane washing and the u se of zwitterionic detergent resulted in the resolution of several integral proteins and the disappearance of peripheral proteins. In the resolution o f expressed genome proteins, both large pH gradients in the first dimension and various acrylamide concentrations in the second dimension must be used . Notwithstanding, it is important to combine various sample treatments and different detergents in order to resolve soluble and hydrophobic proteins.