More than 25 years ago, O'Farrell [O'Farrell P H. J. Biol. Chem. 1975, 250:4007–4021] published a method for high-resolution separation of proteins of the bacteria Escherichia coli using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). In the meantime, this method has been refined and today it is one of the most applied techniques for the analysis and characterization of complex protein mixtures.
The application of isoelectric focusing (IEF) as the first step of 2-D PAGE allows the separation of the proteins on the basis of their charge, and may be performed in polyacrylamide gels with or without an immobilized pH gradient [cf. Görg A., Postel W., and Günther S. The current state of 2-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 1988, 9:531–546]. In the second step, polyacrylamide gels, which contain sodium dodecyl sulfate (SDS) as an anionic detergent and which are particularly suitable for separating proteins on the basis of their molecular weight, are preferably used. Therefore, 2-D PAGE is capable of separating proteins on the basis of two independent parameters, charge and size.
A device for rehydrating a gel strip and performing an IEF as a first step of a 2-D PAGE is known from U.S. Pat. No. 6,113,766. The device includes a chamber which is suitable both for rehydrating a prefabricated and dried gel strip and for performing the IEF. For this purpose, the gel strip is placed in the chamber in such way that—gel side down—each of its end regions comes to rest on one electrode in the chamber floor. The chamber is sealable using a cover, which exercises a specific pressure on the gel strip via pressure parts, so that the gel is pressed onto the electrodes. Following the IEF, i.e., the separation of the proteins in a first dimension, the gel strip is removed from the chamber and laid on an SDS-polyacrylamide gel for performing the separation of the proteins in the second dimension. The gel strip may be damaged as this is done, which may endanger the success of the entire 2-D gel electrophoresis. In addition, achieving a pressure which is sufficiently large to ensure the electrical contact for the IEF, but is small enough that the gel is not damaged is extremely difficult and complicated, because the degree of rehydration of the IEF gel additionally influences its volume.
A solution of the first problem described is known from German Patent 198 31 210, in which a practically simultaneous casting of the gel for the first and second dimension in a joint device is disclosed. The IEF gel is only separated from the SDS-PAGE gel by a narrow element, which may be removed after completion of the IEF and thus leaves a space open which may be filled with a contact gel to bring both gels into contact. The SDS-PAGE may be performed after this. This solution has the advantage that the IEF gel strip does not have to be touched or transported at all between the first and second dimension of a 2-D PAGE. However, it is disadvantageous that both gels must be discarded if the IEF is not successful. In addition, it is known that the reproducibility of IEF results is significantly improved if IEF gels of the same batch are used. This would mean that a large number of gels for the first and second dimension would have to be cast at the same time and under the same conditions, which may become very costly.
Another solution of the first problem described is disclosed in U.S. Pat. No. 5,993,627. In a fully automated system for performing 2-D gel electrophoresis, gels for both the first and the second dimension are cast. The system also includes devices for performing the electrophoresis, the subsequent gel staining, and the analysis. The system is based on the production of IEF gels on a “backing material” made of Gelbond®, from the transfer of this gel into a mold for casting the SDS gel, into which a massive electrode is also cast simultaneously. The system requires multiple robot arms and/or gripping tools for grasping and transporting the gels from one container (IEF chamber) to the other (SDS-PAGE chamber, staining chamber, and scanning bed). The Gelbond® material does improve the stability of the IEF strip, but the method suggested is complicated and costly, and the system is correspondingly expensive.
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