Two‐dimensional electrophoresis with cationic detergents, a powerful tool for the proteomic analysis of myelin proteins. Part 1: Technical aspects of electrophoresis

Abstract

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.

L-MPZ, a Novel Isoform of Myelin P0, Is Produced by Stop Codon Readthrough

“…All procedures were carried out on ice or at 4°C. Homogenates were obtained as described (18) with slight modification. Briefly, ScNs were dissected and snap frozen in liquid nitrogen.…”

“…Western Blot Analysis-Western blot analysis using SDS-PAGE and two-dimensional gel electrophoresis (2DE) with a cationic detergent, cetyltrimethylammonium bromide (CTAB), was performed as described (18). This 2DE method using cationic detergent is appropriate for the analysis of highly hydrophobic membrane proteins, especially those in myelin (18,19).…”

“…This 2DE method using cationic detergent is appropriate for the analysis of highly hydrophobic membrane proteins, especially those in myelin (18,19). CTAB-2DE is advantageous for the solubilization of hydrophobic proteins.…”

“…MS Analysis-PNS myelin proteins were separated with CTAB-2DE and visualized with fluorescent staining (18,21). The excised 36-kDa spot was analyzed by the Pasarow Mass Spectrometry Laboratory at the University of California at Los Angeles.…”

“…spots (18). Proteins from rat ScN homogenates were visualized with silver staining of CTAB-2DE membranes (left panels of Fig.…”