Extraction of yeast mitochondrial membrane proteins by solubilization and detergent/polymer aqueous two-phase partitioning.
Identification and characterization of membrane proteins is of increasing importance in modern proteomic studies. It is of central interest to have access to methods that combine efficient solubilization with enrichment of proteins and intact protein complexes. Separation methods have been developed based on nondenaturing detergent extraction of yeast mitochondrial membrane proteins followed by enrichment of hydrophobic proteins in aqueous two-phase system. Combining the zwitterionic detergent Zwittergent 3-10 and the nonionic detergent Triton X-114 results in a complementary solubilization of proteins, which is similar to that of the anionic detergent sodium dodecyl sulfate (SDS) but with the important advantage of being nondenaturing. Detergent/polymer two-phase system partitioning offers removal of soluble proteins, which can be further improved by manipulation of the driving forces governing protein distribution between the phases. Integral and peripheral membrane protein subunits from intact membrane protein complexes partition to the detergent phase while soluble proteins are found in the polymer phase. A protocol is presented which combines nondenaturing solubilization of membrane proteins with extraction in detergent/polymer two-phase system for application in proteomic studies as a mild and efficient method for enrichment of membrane proteins and membrane protein complexes.
Everberg
Henrik
H
Department of Biochemistry, Lund University, Luund, Sweden.
Gustavsson
Niklas
N
Tjerneld
Folke
F
D005591:Chemical Fractionation / Q000379:methods*; D003902:Detergents / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D057927:Hydrophobic and Hydrophilic Interactions; D024101:Mitochondrial Proteins / Q000302:isolation & purification*; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000737:chemistry; D011108:Polymers / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry
D003902:Detergents; D024101:Mitochondrial Proteins; D011108:Polymers; D000644:Quaternary Ammonium Compounds; D029701:Saccharomyces cerevisiae Proteins; C034730:zwittergent 3-12; D012967:Sodium Dodecyl Sulfate; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40