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SDS-PAGE is a fundamental method for 2D map analysis, because as it represents the second dimension run and the run that will finally remain in the record, since it

FIGURE 2.2. Pictogram of the "focusing" of protein-SDS complexes along a PAG containing a gradient of positive charges (in this case 0-18 mM Immobiline pK 10.3). Small proteins/peptides are focused in gel regions of low charge density (i.e., close to the application point), whereas larger proteins migrate progressively down the gel length toward regions of higher positive charge densities until reaching a "pi" due to balancing of positive gel charges with the negative charges of the various SDS-protein complexes.

FIGURE 2.2. Pictogram of the "focusing" of protein-SDS complexes along a PAG containing a gradient of positive charges (in this case 0-18 mM Immobiline pK 10.3). Small proteins/peptides are focused in gel regions of low charge density (i.e., close to the application point), whereas larger proteins migrate progressively down the gel length toward regions of higher positive charge densities until reaching a "pi" due to balancing of positive gel charges with the negative charges of the various SDS-protein complexes.

is at the end of this step that proteins are stained, or blotted and extracted and further analyzed with the powerful tools today available in proteomics, such as MALDI time of flight (MALDI-TOF) MS of both (a) the intact polypeptide chain and (b) its peptides generated by proteolytic digestion. SDS-PAGE seems to have reached a plateau in terms of method development. It is hard to conceive that the technique could be further improved, since just about all possible methodological advances known in electrokinetic methods have been applied here. Yet, new developments appear to be in sight. Zilberstein et al. [18] have just reported a novel method, called "SDS-PAGE focusing," that exploits a "steady-state" process by which the SDS-protein micelles are driven to stationary zones along the migration path against a gradient of positive charges affixed to the neutral polyacrylamide matrix (Figure 2.2). As the total negative surface charge of such complexes matches the surrounding charge density of the matrix, the SDS-protein complex stops migrating and remains stationary, as typical of steady-state separation techniques. As a result of this mechanism, the proteins are separated in an unorthodox way, with the smaller proteins/peptides staying closer to the application point and larger proteins migrating further down toward the anodic gel end. This results in a positive slope of the Mr versus migration plot, versus a negative slope in conventional SDS-PAGE. Particularly advantageous appears the ability of the present method to fine tune the separation of small size fragments and tryptic digests, where conventional SDS-PAGE usually fails. Additionally, by exploiting constant plateaus of charges, rather than gradients, it is possible to amplify the separation between species having closely spaced M r values, down to a limit of ~150 Da. This increments the resolution by at least one order of magnitude as compared with standard

SDS-PAGE, where for a proper separation of two adjacent species, an Mr increment of ~3000 Da is needed.

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