NEpHGE and pI Strip Proteomic 2-D Gel Electrophoretic Mapping of Lipid-Rich Membranes
Two-dimensional gel electrophoresis (2-DE) has become a powerful and widely used technique for proteomic analyses. However, proteins that are highly basic (pI 8–12), suggesting interactions with acidic sulfo- and phospholipids (1), can become compacted at the edge of the gel. Even if the isoelectric focusing (IEF) pH gradient is extended to high pH, slightly basic proteins enter the gel but are not well resolved, and the pH gradient is not sufficiently extended to include highly basic proteins. Therefore, the use of nonequilibrium pH gradient electrophoresis (NEpHGE) may be recommended (1, 2, 3) (see Note 1). In the first dimension, positively charged, basic proteins move toward the negative end of the gel while the pI gradient is forming. Because running the gel to equilibrium would result in many highly basic proteins exiting from the basic end of the gel (negative electrode), the electrophoresis must be stopped at a critical point (thus, “nonequilibrium”).
KeywordsGlycerol Microwave Urea Glycine Sedimentation
- 3.Celis, J. E., Ratz, G., Basse, B., Lauridsen, J. B., and Celis, A. High resolution two-dimensional gel electrophoresis of proteins: isoelectric focusing (IEF) and non equilibrium pH gradient electrophoresis (NEPHGE). Internet web site: http://proteomics.concer.dk/
- 7.Marta, C. B., Taylor, C. M., Cheng S., Quarles, R., Bansal, R., and Pfeiffer, S. E. (2004) Myelin associated glycoprotein cross-linking triggers its partitioning into lipid rafts, specific signaling events and cytoskeletal rearrangements in oligodendrocytes. Neuron Glia Biology 1, 35–46.PubMedCrossRefGoogle Scholar
- 9.Mozdzanowski, J., Speicher, D., and Harper, S. (1995) Two-dimensional electrophoresis. In:Colligan, J. E., Dunn, B. M., Ploegh, H. L., Speicher, D. W., and Wingfield, P. T. (eds), Current Protocols in Protein Science. New York: John Wiley amp; Sons, pp. 10.4.1–30.Google Scholar