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 proteins from Arabidopsis thaliana leaf plasma membrane (PM) model plants, prior to analysis on 2-DE immobilized pH gradient (IPG) gels. Solubilization efficiency of two detergents, (3-[(3-cholomidopropyl)-1-propanesulfonic acid (CHAPS) and C8phi, were tested for the recovery of hydrophobic proteins. An immunological approach was used to determine the efficiency of the above methods. Fractionation of proteins by Triton X-114 combined with solubilization with CHAPS resulted in the inability to detect hydrophobic proteins on 2-DE gels. The use of C8phi for protein solubilization did not improve this result. On the contrary, after treatment of membranes with alkaline buffer, the solubilization of PM proteins with detergent C8phi permitted the recovery of such proteins on 2-DE gels. The combination of membrane washing and the use of zwitterionic detergent resulted in the resolution of several integral proteins and the disappearance of peripheral proteins. In the resolution of 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.
Santoni
V
V
Biochimie et Physiologie Moléculaire des Plantes, INRA/ENSA-M/CNRS URA 2133, Montpellier, France. santoniv@ensam.inra.fr
Rabilloud
T
T
Doumas
P
P
Rouquié
D
D
Mansion
M
M
Kieffer
S
S
Garin
J
J
Rossignol
M
M
D000180:Acrylic Resins; D017360:Arabidopsis / Q000737:chemistry; D002254:Carbonates; D002458:Cell Fractionation; D002793:Cholic Acids; D003902:Detergents; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D017830:Octoxynol; D018515:Plant Leaves / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011092:Polyethylene Glycols; D012995:Solubility
D000180:Acrylic Resins; D002254:Carbonates; D002793:Cholic Acids; D003902:Detergents; D010940:Plant Proteins; C016680:polyacrylamide gels; D011092:Polyethylene Glycols; C005686:sodium carbonate; D017830:Octoxynol; C010615:Nonidet P-40; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate