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Two‐dimensional electrophoresis with cationic detergents, a powerful tool for the proteomic analysis of myelin proteins. Part 1: Technical aspects of electrophoresis
Author(s) -
Yamaguchi Yoshihide,
Miyagi Yudai,
Baba Hiroko
Publication year - 2008
Publication title -
journal of neuroscience research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.72
H-Index - 160
eISSN - 1097-4547
pISSN - 0360-4012
DOI - 10.1002/jnr.21547
Subject(s) - isoelectric focusing , myelin , cationic polymerization , polyacrylamide gel electrophoresis , gel electrophoresis , chromatography , electrophoresis , sodium dodecyl sulfate , chemistry , isoelectric point , myelin basic protein , biochemistry , biology , organic chemistry , enzyme , neuroscience , central nervous system
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. © 2007 Wiley‐Liss, Inc.