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Before separation, proteins of different biological samples are labeled with different fluorescent dyes, the CyDye DIGE Fluors. Currently three dyes with spectrally different excitation and emission wavelengths are available. This allows labeling up to three different samples, and coseparating them in one gel. The dyes can either be attached to the epsilon-amino side group of the lysine without derivatization of the polypeptides or to the cysteines after reduction of the disulfide bonds. For lysine labeling a so called minimal labeling approach is performed: only a low-ratio dye: protein is applied in order to prevent multiple labels per protein. Although only 3% of the proteins are tagged, the sensitivity of detection is comparable with the sensitivity of a good quality silver staining. The dyes are matched for size and charge to obtain migration of differently labeled identical proteins to the same spot positions. The spot pattern achieved with minimal labeling is similar to the pattern obtained with poststained gels. When cysteine tagging is applied, all cysteine moieties are labeled. This modification of the method affords extraordinarily high sensitivity of detection. However, because of multiple labeling, the resulting pattern will look different from nonlabeled or minimal labeled samples. The labeled samples are mixed together before they are applied on the gel of the first dimension. After separation the gels are scanned with the multifluorescent imager at the different wavelengths. Up to three images of comigrated protein mixtures are compared and evaluated from each gel. This multiplexing technique allows the application of an internal standard for each protein in a complex mixture: One of the labels is applied on a mixture of the pooled aliquots of all samples of an experiment. By coseparating this mixture with each gel an internal standard is created for reliable and reproducible detection and assessment of changes of protein expression levels. Image analysis is performed with special software, which allows codetection of protein spots across the different samples and the internal standard.

In spite of text books, instrument manuals, product instructions, and web tutorials there are a number of erroneous protocols around, which lead repeatedly to issues during electrophoresis runs and to inadequate results. The relatively low resolution and short running time of miniformat systems often conceals these issues. However, in high-resolution 2-D electrophoresis in large format gels, one of the most important separation methods in Proteomics, the consequences of these mistakes become more obvious.

Publisher Summary Isoelectric focusing (IEF) is most commonly carried out in polyacrylamide slab gels. Over the years, the slab format has proved preferable to other possible configurations for the technique. The first practical realization of IEF was performed in glass columns containing sucrose density gradients. The pH gradients were generated with carrier ampholytes and the sucrose density gradient stabilized focused bands against precipitation. This chapter presents the first apparatus for performing IEF in polyacrylamide slab gels. The chapter describes the most successful approaches to slab gel IEF. The phenomenon called “electroendosmosis” can destroy a pH gradient and ruin resolution. It occurs when the separation matrix and (or) parts of the equipment that are in direct contact with the gel contain fixed charges. Polyacrylamide is an ideal matrix for IEF with both carrier ampholytes and immobilized pH gradients. It is chemically inert, compact, completely transparent, and free of electroendosmosis. Some laboratories use vertical slab gel electrophoresis equipment for IEF. In this case, the gel remains in the casting cassette between two glass plates during the run. There are two ways to polymerize polyacrylamide gels: (1) chemical polymerization with tetramethylethylenediamine (TEMED) and ammonium persulfate; and (2) photopolymerization with riboflavin and TEMED.

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).

Using polyacrylamide electrode gels during horizontal sodium lauryl sulfate-polyacrylamide electrophoresis of radiolabeled proteins instead of electrode papers reaching into electrode buffer reservoirs, silver staining is improved and we reduce chemical and radioactive liquid waste during electrophoresis.

Buffer-soluble seed proteins of 12 varieties of winged bean (Psophocarpus tetragonolobus L.) were separated by ultrathin-layer isoelectric focusing (IEF) and sodium dodecylsulphate electrophoresis(SDS-PAGE). Whereas the molecular weight spectra of the proteins obtained by SDS-PAGE were identical for all varieties studied, the IEF patterns showed variety specificity for each sample. Most of the variety specific protein bands were identified as glycoproteins. The zymograms of the isoelectically focused trypsin inhibitors were variety specific in both activity and isoelectric point distribution.

A new method for electrophoretic retrieval of protein zones from Immobiline matrices is described, based on elution directly in a free liquid phase, rather than in ion-exchange beads or molecular sieves, as previously described. The chopped Immobiline gel is loaded on top of a 5% T stacking gel, 6-10 mm in height, and forced to transverse it and collect into a chamber, filled with 20% sucrose solution, closed on its anodic side by a dialysis sac. The transfer is practically quantitative, for most proteins, after 30-60 min of zone electrophoresis at 10 W (300 V potential differential). Recovery of protein mass is in general better than 90%, while for enzyme activity is in the range of 60-80%. For preserving enzyme integrity, the following precautions are recommended: short electrophoretic times; avoidance of anodic oxidation; chilling of the buffer in the anodic chamber; and use of low levels (2-5 mM) of the specific enzyme substrate throughout the entire electrophoretic system (cathode, anode and gel plug).

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.

A method of ultrathin-layer isoelectric focusing in 0.12-, 0.24-, or 0.36-mm polyacrylamide gel layers polymerized on a sheet of cellophane as support is deseribed. The gel adheres firmly to the cellophane during all operation steps, is protected from fracture, and can be handied very conveniently. Resolution is markedly improved in ultrathin gels in comparison with the conventional 1- to 2-mm-thick gels. Staining and destaining are completed in a substantially shorter time than so far achieved. The ultrathin gels can be easily dried on the cellophane, a perfectly transparent record being obtained for future reference and for densitometric evaluation. Results are presented for a number of commercial proteins and legume seed proteins. The advantages of ultrathin-layer isoelectric focusing are discussed.

A new technique for generating pH gradients in isoelectric focusing is described, based on the principle that the buffering groups are covalently linked to the matrix used as anticonvective medium. For the generation of this type of pH gradient in polyacrylamide gels, a set of buffering monomers, called Immobiline (in analogy with Ampholine), is used. The pH gradient gels are cast in the same way as pore gradient gels, but instead of varying the acrylamide content, the light and heavy solutions are adjusted to different pH values with the aid of the Immobiline buffers. Available Immobiline species make it possible to generate any narrow linear pH gradient between pH 3 and 10. The behaviour of these types of gradients in isoelectric focusing is described. Immobilized pH gradients show a number of advantages compared with carrier ampholyte generated pH gradients. The most important are: (1) the cathodic drift is completely abolished; (2) they give higher resolution and higher loading capacity; (3) they have uniform conductivity and buffering capacity; (4) they represent a milieu of known and controlled ionic strength.

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.