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In proteomics research chemical as well as physical methods are used to detect proteins subsequently to their separation. Physical methods are mostly applied after chromatography. They are either based on spectroscopy like light absorption at certain wavelengths or mass determination of peptides and their fragments with mass spectrometry. Chemical methods are used after two-dimensional electrophoresis and employ staining with organic dyes, metal chelates, fluorescent dyes, complexing with silver, or pre-labeling with fluorophores. In some cases autoradiography is still used. Since all of these techniques are very different in terms of sensitivity, their usefulness for quantitative determinations varies significantly. This review will describe the various protein detection methods applied to electrophoresis gels.

Fluorescent pre-labelling of proteins prior to electrophoretic separations is not only advantageous for multiplex detection strategies, like DIGE (difference gel electrophoresis), but also as a general protein visualization procedure. We introduce a fluorescent amino-reactive dye which is compatible with all standard additives used for protein sample preparation including reductants. Fluorescent pre-labelling is less time-consuming and as sensitive as silver staining with additionally excellent quantification properties.

Die 2D-Elektrophorese von intakten Proteinen ist die zentrale Trennmethode der top-down-Strategie in der Proteomanalytik. Hierfur sollten alle technischen Moglichkeiten zur Optimierung der Trennergebnisse und Reproduzierbarkeit ausgeschogft werden.

Today the omnipresence of MS-based proteomic approaches suggest, that 2-dimensional gel (2-DE) based proteomics is dead. In reality, this is far from the truth, and also after three decades high-resolution 2-DE, introduced by Patrick O’Farrell in 1975, still represents a key technology to investigate thousands of protein species, their isoforms and modifications simultaneously. Especially, in combination with multi fluorescence labelling and various MS-methodologies, it is very popular to relatively quantify and identify proteins in complex mixtures. But also after these 30 years of use, reproducibility and process stability are still under constant improvement to further enhance robustness and feasibility of the method. In order to offer an opportunity to discuss and apply the latest developments in 2-DE in a scientific context we picked out this technology as central theme for the International Protein Rainbow Workshop titled ‘‘2-Dimensional Gel Electrophoresis Reloaded’’, which was held at the Institute of Clinical Biochemistry and Pathobiochemistry at the German Diabetes-Center in Duesseldorf. This annual hands-on training course, comprising theoretical and practical aspects of the technology, was accompanied by a lecture day giving an overview of recent 2-DE applications in scientific research. In dependence on this the current issue ‘‘2-Dimensional Gel Electrophoresis Reloaded’’ should help to give an overview of recent developments and applications related to 2-DE. The collection of scientific reports covers a broad range of applications, starting with an inter-laboratory comparison introducing a simplified and robust 2-DE work-flow. In addition a novel fluorescent dye for improved protein detection is described and pre-analytical conditions for serum proteomics, representing one crucial point for biomarker discovery are addressed. Another study examines differences and similarities between protein patterns derived from mitochondria and peroxisomes or describe the characterization of sub-types in small cell lung cancer (SCLC).

Horizontal SDS electrophoresis of 18 legume seed protein extracts was performed in ultrathin-layer polyacrylamide gels on foil supports. Separation results of the SD S pore gradient electrophoresis (T=4–22.5%) are compared to those of SDS electrophoresis in a constant pore size gel (T=10%). Resolution as well as the sensitivity (0.1 μg protein per band) of the ultrathin-layer SDS pore-gradient electrophoresis were extremely high. Because of the very low gel thickness, separation, staining and drying were completed in substantially shorter times than achieved with conventional thick gels. An easy technique for casting ultrathin-layer (360 gm) concave gradient gels for 10 cm separation distance and a width of 25 cm is described. The even distribution of the concave exponential pore-gradient over the whole gel width is demonstrated. Molecular weights of the legume proteins are detected from 5,000 to 110,000 daltons. The protein patterns are genus- and species-specific.

Legume seed proteins extracted with urea-containing buffer are fractionated by high-resolution 2D-electrophoresis (urea-IEF x pore gradient SDS-PAGE), both dimensions in horizontal ultrathin-layer polyacrylamide gel slabs. High reproducibility is obtained, because the first dimension is performed in a slab gel, where a large number of protein samples are separated under identical conditions. The gel of the first dimension (IEF) is fixed, stained with Coomassie Brilliant Blue G 250, and destained before application to the second-dimension gel (SDS-PAGE). Prestaining of the focused proteins does not alter the protein pattern obtained after SDS electrophoresis. Thus, bands are made visible before separation in the second dimension, and the amount of Ampholine in the dye front is reduced during electrophoresis. The first-dimension gels can easily be stored in the destaining solution until they are run in the second dimension. As the proteins are fixed in the gel, there is no loss of proteins and defocusing of bands due to diffusion during the SDS-equilibration procedure. Loading of the dimensionstable gel strip onto the second dimension gel is a very easy operation, since the ultrathin gel strip adheres to a plastic foil and is simply laid into a moulded gel through. The gel strip is not imbedded with polymerizing acrylamide or agarose solution like in the conventional gel-rod-techniques, because a very good surface contact is obtained between the two flat gels.

The human serum transferrin (Tf) system was analyzed by isoelectric focusing (IEF) with immobilized pH gradients. For the demonstration of the genetic variability the Fe1-Tf region was chosen. The pH range suitable for analysis of the Tf system was pH 5.20 to pH 5.75. The phenotypes of the common six TfC subtypes are described. No further heterogeneity among TfC1, TfC2, and TfC3 was noted. Also presented are the phenotypes of the TfC6 subtype, and of three different TfB and three different TfD variants. IEF with immobilized pH gradients appears to be a suitable method for the analysis of the inherited transferrin polymorphism.