Two-dimensional electrophoresis with cationic detergents, a powerful tool for the proteomic analysis of myelin proteins. Part 1: technical aspects of electrophoresis.
The analysis of proteins in damaged myelin is crucial to clarify the mechanisms of dysmyelination and demyelination. In the present study, proteomic analysis of myelin using a modified two-dimensional electrophoresis (2-DE) method was carried out to obtain a better understanding of myelin biology. Although standard 2-DE (immobilized pH gradient isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis; IPG/SDS-PAGE) methods of analysis provide high resolutions of soluble proteins with isoelectric focusing points in the pH range of 4-8, major myelin components include highly basic proteins are compacted at the basic edge of the 2-DE gels and are not sufficiently separated for satisfactory analysis. In an attempt to improve the separation of these proteins, an alternative 2-DE method using the cationic detergents was applied. In part 1 of this study, we describe technical aspects of conditioning 2-DE using cationic detergent. In the accompanying paper (part 2), practical 2-DE analysis using cationic detergents is described to identify proteins in the purified CNS myelin fraction. We carried out benzyldimethyl-n-hexadecylammonium chloride (16-BAC)/SDS-PAGE 2-DE and tested 2-DE with four other cationic detergents. We found that 16-BAC was the most effective agent for separation of myelin proteins and that hexadecyltrimethylammonium bromide (cetyltrimethylammonium bromide; CTAB) was the most effective agent for solubilization of myelin proteins. The combination of 16-BAC/SDS-PAGE and CTAB/SDS-PAGE is a powerful tool for the analysis of myelin proteins, including highly basic, high-MW (MW > 100K), and integral membrane proteins.
Yamaguchi
Yoshihide
Y
Department of Molecular Neurobiology, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan. yoshiy@ps.toyaku.ac.jp
Miyagi
Yudai
Y
Baba
Hiroko
H
D000818:Animals; D015153:Blotting, Western; D001923:Brain Chemistry; D002412:Cations; D003902:Detergents; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D009185:Myelin Proteins / Q000737:chemistry* / Q000302:isolation & purification; D009186:Myelin Sheath / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D051381:Rats
D002412:Cations; D003902:Detergents; D009185:Myelin Proteins