Highly basic myelin and oligodendrocyte proteins analyzed by NEPHGE-two-dimensional gel electrophoresis: Recognition of novel developmentally regulated proteins

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) provides high resolution separation of proteins and offers a powerful method for their id entification and characterization. Since many myelin-specific proteins are highly basic, they cannot readily be analyzed by standard isoelectric focus ing (IEF)2D-PAGE that affords separation primarily in the isoelectric point s (pI) range of 4-8. An alternative method, nonequilibrium pH gradient elec trophoresis (NEPHGE)-2D-PAGE, can provide excellent resolution of highly ba sic proteins. In the present study, we have optimized the NEPHGE-2D-PAGE pr otocol for the analysis of myelin proteins with basic pIs, and provide a NE PHGE-2D-PAGE map based on size, pI, and immunoreactivity (Western blot) of myelin basic protein (MBP), 2',3'-cyclic-nucleotide 3'-phosphodiesterase (C NP), myelin proteolipid protein (PLP), and its smaller spliced variant DM20 , myelin/oligodendrocyte glycoprotein (MOG) and oligodendrocyte-specific pr otein (OSP), We have also demonstrated, by analyzing metabolically radiolab eled oligodendrocytes in culture at specific stages of the developmental li neage, the developmentally up-regulated expressions of several undefined, o ligodendrocyte, basic membrane proteins during oligodendrocyte differentiat ion. We suggest that this approach offers an important tool for identifying and characterizing the plethora of uncharacterized myelin proteins. J. Neu rosci. Res. 56:199-205, 1999, (C) 1999 Wiley-Liss, Inc.