b'1D vs. 2D PAGE Protein gel chemistriesThe most common form of protein gel electrophoresisPAGE utilizes a discontinuous buffer system to concentrate is comparative analysis of multiple samples byor stack samples into a very sharp zone in the stacking one-dimensional (1D) electrophoresis, in which samplesgel at the beginning of the run. In a discontinuous buffer are loaded into wells, a current is applied to separate thesystem, the primary anion in the gel is different (or proteins, and the resulting migration of the protein bands isdiscontinuous) from the primary anion in the running buffer. visualized by staining or on a western blot. Invitrogen Bolt Bis-Tris Plus gels, Invitrogen NuPAGE Bis-Tris and Tris-Acetate gels, and the Laemmli systemMultiple components of a single sample can be resolvedbased Invitrogen Novex Tris-Glycine gels are examples of most completely by two-dimensional electrophoresisdiscontinuous buffer systems and work in a similar fashion. (2D-PAGE). The first dimension separates proteinsHowever, Bis-Tris and Tris-acetate systems operate at a according to their native isoelectric point (pI) using a formlower pH as a result of the ions that are in the system.of electrophoresis called isoelectric focusing (IEF). The second dimension separates proteins by mass usingTris-glycine chemistryordinary SDS-PAGE. 2D PAGE provides the highestIn a Tris-glycine system (Figure 1), three ions are resolution for protein analysis and is an important techniqueprimarily involved:in proteomic research, where resolution of thousands of proteins on a single gel is sometimes necessary. The main Chloride (), supplied by the gel buffer, serves as the focus of this handbook is on 1D electrophoresis. leading ion because it has the highest attraction to the anode relative to other anions in the system. Glycine (), the primary anion provided by the running buffer, serves as the trailing ion, because it is only partially negatively charged and remains behind the more highly charged chloride ions in a charged environment. Tris base (+), a common ion present in both the gel and the running buffers. During electrophoresis, the gel and buffer ions in the Tris-glycine system form an operating pH of 9.5 in the separating region of the gel.Bis-Tris chemistryIn the case of the Bis-Tris system (Figure 2), three ions are primarily involved: Chloride () supplied by the gel buffer, serves as the fast-moving leading ion. MES or MOPS () (depending on the running buffer choice) serves as the trailing ion. MES: 2-(N-morpholino) ethane sulfonic acid MOPS: 3-(N-morpholino) propane sulfonic acid Bis-Tris (+) acts as the common ion present in the gel while Tris (+) is provided by the running buffer.6'