Highly basic myelin and oligodendrocyte proteins analyzed by NEPHGE-two-dimensional gel electrophoresis: Recognition of novel developmentally regulated proteins
✍ Scribed by Y. Yamaguchi; S.E. Pfeiffer
- Publisher
- John Wiley and Sons
- Year
- 1999
- Tongue
- English
- Weight
- 102 KB
- Volume
- 56
- Category
- Article
- ISSN
- 0360-4012
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✦ Synopsis
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) provides high resolution separation of proteins and offers a powerful method for their identification and characterization. Since many myelinspecific proteins are highly basic, they cannot readily be analyzed by standard isoelectric focusing (IEF)-2D-PAGE that affords separation primarily in the isoelectric points (pI) range of 4-8. An alternative method, nonequilibrium pH gradient electrophoresis (NEPHGE)-2D-PAGE, can provide excellent resolution of highly basic proteins. In the present study, we have optimized the NEPHGE-2D-PAGE protocol for the analysis of myelin proteins with basic pIs, and provide a NEPHGE-2D-PAGE map based on size, pI, and immunoreactivity (Western blot) of myelin basic protein (MBP), 2Ј,3Ј-cyclic-nucleotide 3Ј-phosphodiesterase (CNP), myelin proteolipid protein (PLP), and its smaller spliced variant DM20, myelin/oligodendrocyte glycoprotein (MOG) and oligodendrocyte-specific protein (OSP). We have also demonstrated, by analyzing metabolically radiolabeled oligodendrocytes in culture at specific stages of the developmental lineage, the developmentally up-regulated expressions of several undefined, oligodendrocyte, basic membrane proteins during oligodendrocyte differentiation. We suggest that this approach offers an important tool for identifying and characterizing the plethora of uncharacterized myelin proteins.