Separation techniques isoelectric focusing

While all of these devices used normal zone electrophoresis separation techniques, isoelectric focusing (IEF) methods on microchips have also been interfaced with ESI-MS for high-resolution separations of proteins [37], Figure 5 shows the electropherogram and corresponding mass spectra generated by this device. For on-chip sample preconcentration before separation, a polarityswitching technique was employed to achieve subnanomolar detection limits for many peptide standards [38],... 

Consider the electrophoretic motion of charged mol-ecules/macromolecules/proteins in an aqueous solution/ buffer subjected to an electrical force. Three separation techniques, isoelectric focusing, capillary electrophoresis... 

Innovations in separation science continued on this theme and provided one of the most powerful separation techniques used in biochemistry, where proteins are separated with isoelectric focusing (IEF) applied in one direction, and gel electrophoresis (GE) applied at aright angle to the first separation direction (O Farrell, 1975 Celis and Bravo, 1984). In this case, proteins are first separated according to their isoelectric point, measured in p/units, and then according to their molecular weight by gel electrophoresis. The size separation step is usually aided by addition of a surfactant, most typically sodium dodecyl sulfate (SDS), and the gel material is a polyacrylamide formulation. 

FIGURE 3-22 Two-dimensional electrophoresis, (a) Proteins are first separated by isoelectric focusing in a cylindrical gel. The gel is then laid horizontally on a second, slab-shaped gel, and the proteins are separated by SDS polyacrylamide gel electrophoresis. Horizontal separation reflects differences in pi vertical separation reflects differences in molecular weight, (b) More than 1,000 different proteins from . coli can be resolved using this technique. 

Isoelectric Focusing. Isoelectric focusing is a technique used for protein separation, by driving proteins to a pH where they have no mobiUty. Resolution depends on the slope of a pH gradient that can be achieved in a gel. 

Two-dimensional gel electrophoresis (2DE) is a two-dimensional technique for protein separation, which combines isoelectric focusing and sodium dodecyl sulphate (SDS) electrophoresis. The high resolving power results from separation according to charge (isoelectric point) in the first dimension and size (mobility in a porous gel) in the second dimension. Depending on the gel size, from several hundred to more than 5,000 proteins can be separated. 

Ruchel, R. (1977). Two-dimensional micro-separation technique for proteins and peptides, combining isoelectric focusing and gel gradient electrophoresis. J. Chromatogr. 132, 451 168. 

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