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The first feasibility studies of three-dimensional (3-D) geometry gel electrophoresis were independently conducted and published by two groups in 2003 (1,2). Common to both described concepts is that the separation medium extends substantially in all three spatial coordinates. The samples are applied in a two-dimensional (2-D) loading plane on top of the 3-D gel body. The electrophoretic separation occurs along the third spatial coordinate, perpendicular to the loading plane.
The team of Bao-Shiang Lee proposed a 3-D gel cube to improve the molecular mass resolution of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) protein analysis (1).
The other feasibility study, published concurrently by the authors of this article, solved the problem of irregular sample migration (smiling) and gel-to-gel variations by a specific heat conduction concept (2). A thermal insulation encircling the 3-D gel (Figure 1) blocks heat transfer in the radial direction, while the top and bottom of the gel are cooled by circulating the buffer through a thermostat. This forces the heat flow parallel to the gel's vertical axis and avoids temperature gradients perpendicular to the sample migration direction. In addition, our first 3-D gel instrument provided a system for online detection of the samples by laser-induced fluorescence (LIF). The fluorescently labeled sample fractions are recorded by a photodetector while passing a laser-illuminated area during electrophoresis, similar to the operation of automated DNA sequencers (3). We proposed and tested several applications, such as DNA sequencing, DNA fragment analysis, and SDS-PAGE protein analysis. This early study demonstrated that 3-D gel electrophoresis could be used for analysis of biomolecules with one, two, or three independent separation parameters.
Examples for one-parameter high-throughput separation are the quality validation of purified proteins prior to crystallization (4) and the analysis of high-performance liquid chromatography (HPLC)-prefractionated proteins by SDSPAGE. The loading in a 2-D array on top of the 3-D gel results in a very high sample capacity in which the density is limited only by lateral diffusion. For efficient loading, we described a method for simultaneous electrokinetic transfer of all samples from 96-, 384-, or 1536-well microtiter plates directly onto the 3-D gel (4).
An example for two-parameter comparative separation is the analysis of protein aggregates by blue native electrophoresis (BNE), followed by SDS-PAGE in the 3-D gel (5). The method is particularly suited for the monitoring of aggregate formation (e.g., in clinical samples) or screening of drug candidates for their effects on protein aggregation, for instance during amyloid-related neurodegenerative disease development. Furthermore, changes in functional protein complex formation are important prognostic characteristics for changes in cellular signaling processes and disease states.
Although not implemented so far, a genuine three-parameter separation is technologically feasible. An example would be the analysis of protein aggregates or complexes by BNE, followed by isoelectric focusing (IEF) in a slab gel, and SDS-PAGE in the 3-D gel for separation and identification of the aggregates’ components.
Among the possible applications of the 3-D gel, we consider comparative IEF/SDS-PAGE protein analysis to constitute the most promising. Conventional 2-D gel electrophoresis (2-DE) analysis is a strenuous and time-consuming procedure, precluding the comparative examination of several tens or hundreds of samples. Moreover, the gel-to-gel variations pertinent to standard 2-DE require elaborate software corrections of the images and the running of a large number of gels to obtain reliable results in comparative studies. Our 3-D gel electrophoresis method inherently supports automated high-throughput 2-DE protein analysis with immediate comparability of the separation patterns. Together with the online detection, this makes the overall analysis considerably more efficient. We foresee many possible applications in molecular discovery, clinical diagnosis, pharmacology, and toxicology, such as large-scale differential protein analysis, protein monitoring during disease development, as well as screening of drug candidates and chemical compounds for their effect on protein expression and macromolecular assemblies.
