How to Bring the “Unseen” Proteome to the Limelight via Electrophoretic Pre-Fractionation Techniques

Abstract

The present review reports a panoply of electrophoretic methods as pre-fractionation tools in proteomic investigations in preparation for mass spectrometry or two-dimensional electrophoresis map analysis. Such electrophoretic pre-fractionation protocols include all those electrokinetic methodologies which are performed in free solution, most of them relying on isoelectric focusing steps (although some approaches based on gels and granulated media are also discussed). Devices associated with electrophoretic separations are multi-chamber apparatuses, such as the multi-compartment electrolyzers equipped with either isoelectric membranes or with isoelectric beads, Off-Gel electrophoresis in a multi-cup device and the Rotofor, an instrument also based on a multi-chamber system but exploiting the conventional technique of carrier-ampholyte-focusing. Other free-flow systems, as well as miniaturized chambers, are also described.

Coomassie Blue as a Near-infrared Fluorescent Stain: A Systematic Comparison With Sypro Ruby for In-gel Protein Detection

“…Many critical components in normal physiology and also disease may be several orders of magnitude less abundant and thus below the detection threshold of in-gel stains, or indeed most techniques. Pre-and post-fractionation technologies have been developed to address this central issue in proteomics but these are not without limitations (1)(2)(3)(4)(5). Thus improved detection methods for gel-based proteomics continue to be a high priority, and the literature is rich with different in-gel detection methods and innovative improvements (6 -34).…”

Adsorbed protein detection by scanning electrochemical microscopy

“…In systems such as yeast, these proteins furthermore represent 80% of the predicted proteome [3,4]. In gels or in human fingerprints, proteins are usually visualized by optical techniques based on protein staining with either metal ions such as silver [5], organic substances [6] or fluorescent dyes [7][8][9].…”

Protein separation using preparative‐scale dynamic field gradient focusing

“…2, to all other preparative electrophoresis instruments would require a review beyond the scope of this report. Two reviews of commercially available preparative electrophoresis equipment were written by Righetti and co-workers [10,11]. The reviews do not compare the noncommercial vortex-stabilized electrophoresis cham- Figure 1.…”

“…Two other papers [12,13] do mention how the vortex-stabilized electrophoresis chamber differs from earlier preparative electrophoresis instruments. We draw from references [4,5,[10][11][12][13] to briefly illustrate the primary differences between the preparative-scale DFGF chamber and other preparative electrophoresis instruments.…”