Immunoisolation and subfractionation of synaptic vesicle proteins.
Within recent years, the advances in proteomics techniques have resulted in considerable novel insights into the protein expression patterns of specific tissues, cells, and organelles. The information acquired from large-scale proteomics approaches indicated, however, that the proteomic analysis of whole cells or tissues is often not suited to fully unravel the proteomes of individual organellar constituents or to identify proteins that are present at low copy numbers. In addition, the identification of hydrophobic proteins is still a challenge. Therefore, the development of techniques applicable for the enrichment of low-abundance membrane proteins is essential for a comprehensive proteomic analysis. In addition to the enrichment of particular subcellular structures by subcellular fractionation, the spectrum of techniques applicable for proteomics research can be extended toward the separation of integral and peripheral membrane proteins using organic solvents, detergents, and detergent-based aqueous two-phase systems with water-soluble polymers. Here, we discuss the efficacy of a number of experimental protocols. We demonstrate that the appropriate selection of physicochemical conditions results in the isolation of synaptic vesicles of high purity whose proteome can be subfractionated into integral membrane proteins and soluble proteins by several phase separation techniques.
Burré
Jacqueline
J
Department of Cell Biology and Neuroscience, Neurochemistry, JW Goethe-University, Max-von-Laue-Str 9, 60438 Frankfurt, Germany. j.burre@bio.uni-frankfurt.de
Zimmermann
Herbert
H
Volknandt
Walter
W
D000818:Animals; D015153:Blotting, Western; D002725:Chloroform / Q000737:chemistry; D003902:Detergents / Q000737:chemistry; D004072:Digitonin / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D000432:Methanol / Q000737:chemistry; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000737:chemistry; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D051381:Rats; D012997:Solvents / Q000737:chemistry; D055672:Static Electricity; D013572:Synaptic Vesicles / Q000737:chemistry*
D003902:Detergents; D008565:Membrane Proteins; D011506:Proteins; D012997:Solvents; D011092:Polyethylene Glycols; D002725:Chloroform; D017830:Octoxynol; C010615:Nonidet P-40; D004072:Digitonin; D000432:Methanol