Applications

With its two separation modes (1D or 2D separation) and several distinct advantages over conventional capillary and slab gel electrophoresis, the 3D-gel method lends itself to a wide range of applications in different fields and disciplines: 

1D separation

Examples for 1D high-throughput separation are the quality validation of purified proteins prior to crystallisation [Electrophoresis 2006, 27:3338-48] and the analysis of HPLC- prefractionated proteins by SDS-PAGE. The latest instrument prototype is designed for the parallel separation of all samples of a microtiterplate. For efficient loading, all samples are transferred simultaneously from 96-, 384-, or 1536-well microtiterplates directly to the 3D-gel. For visualization, band detection and statistical analysis of the images, we use LabImage 1D software (Kapelan Bio-Imaging, Leipzig, Germany).

A very promising new application for 1D separation is the profiling of binding profiles of small compounds in affinity-based screens. The possibility to separate and identify affinity-captured GFP-tagged fusion proteins by their mass in 3D-gel SDS-PAGE increases the throughput (96, 384 or 1536 extracts pools per gel) such that the screening of an entire proteome in only a few gels becomes feasible.

2D separation

The 3D-gel may also be used for high-throughput analysis with two separation parameters, e.g., comparative analysis of protein aggregates by blue native electrophoresis (BNE) and subsequent SDS-PAGE [Proteomics 2005, 5:2002-09]. Several neurodegenerative diseases involve amyloid-like protein aggregation. Our technique could provide a diagnostic picture during their development and allows the screening of drug candidates for their effects on protein aggregation.

We have also extended the method to comparative 2-DE protein analysis [BioTechniques 2007, 42:271-79]: A plastic frame that holds 36 IPG-strips containing IEF-prefractionated samples, is placed on the 3D-gel and the samples are transferred directly into the gel. For optimum resolution and dynamic range, samples are fluorescently labeled before IEF in only a few steps, e.g., following standard DIGE labeling protocols. The identical electrophoretic and thermal conditions in the 3D-gel permit large-scale comparative 2-DE analyses, e.g., for clinical diagnosis, pharmacology, molecular toxicology, drug interaction analysis and protein expression studies. For visualization, spot detection and statistical analysis of the images, we use Delta2D software (Decodon, Greifswald, Germany).

With the new European Union directive for the registration, evaluation, and authorization of chemicals (REACH), a thorough investigation of substances for their potentially hazardous effects on organisms will become mandatory for their producers. Here, the 3D-gel method could be used for studying changes in protein expression in response to those chemicals.