2‐DE with IPGs

Abstract

In order to overcome the limitations of carrier ampholyte generated pH gradients, IPGs were developed in the late 1970s. However, the 2-DE pattern we included in the first publication on IEF with IPGs [Bjellqvist et al., J. Biochem. Biophys. Methods 1982, 6, 317-339] was far from being competitive to O'Farrell's high-resolution 2-DE with carrier ampholytes. Our 2-DE pattern in this article was, more or less, only a proof of principle. It was, however, the beginning of a long journey of stepwise improved 2-DE protocols we developed in our laboratory and summarized in the reviews published in Electrophoresis 1988, 9, 531-546 and in Electrophoresis 2000, 21, 1037-1053. Milestones were the design of the IPG strip, and the "reduction-alkylation equilibration protocol" of IPG strips after IEF for the efficient transfer of proteins from first to second dimension. The protocol of 2-DE with IPGs has been constantly refined, e.g. by the generation of tailor-made IPGs with different pH intervals from the acidic to the basic extremes (pH 2.5-12), and extended separation distances for improved resolution. In the present review, a historical outline from the technical difficulties encountered during the development of 2-DE with IPGs, to the establishment of the actual "standard protocol" will be given, as well as the modified procedures for the separation of very acidic, very alkaline, low-abundance and hydrophobic proteins, followed by a brief discussion of the advantages and technical challenges of gel-based proteomic technologies.

Proteomic studies in breast cancer (Review)

“…The direction of the protein movement at the second phase is perpendicular to the first, in order to provide a spot map of the proteins distributed in the two dimensions. The 2-DE-separated proteins are detected using several pre-or post-electrophoresis staining or fluorescence techniques (8). In the traditional method of 2-DE, proteins are separated from a complex mixture according to their electrical charge and dimensional differences.…”

The Role of Proteomics in Elucidating Multiple Antibiotic Resistance in Salmonella and in Novel Antibacterial Discovery

“…2-DE gels will probably remain the ''gold standard'' within the foreseeable future to which any competing method should be compared, and to which it should display clear advantages of 2-DE with IPGs (Görg et al, 2009). In contrast to the 2-DE approaches, information about protein abundances is initially unavailable in the nongel-based technologies, unless stable isotope labelling is applied.…”

“…However, after the introduction of large-scale genome sequencing, the development of MS methods for the analysis of proteins and peptides, progress in bioinformatic tools and the rise of proteomics in general has experienced a revival and is today by far the most commonly applied protein separation technology in proteome research (Görg et al, 2009). Recent advances in biological and analytical sciences have led to an unprecedented interest in the discovery and quantification of endogenous molecules which serve as indicators of drug safety, mechanism of action, efficacy, and disease state progression.…”

Two-Dimensional Gel Electrophoresis (2-DE)

“…Narrow pH gradients (such as 4-5, 4.5-5.5, and even in 0.2 pH units with pH 4.35-4.55 gradient) can be overlapped increasing the number of detected spots (Fig. 3); they are well applied to micro-preparative separations with sample loads of up to several milligrams (Westermeier et al, 1983;Görg et al, 2009). …”

“…Exceptionally, the tissue is boiled for 5 minutes in 1-2% SDS before they are diluted with lysis buffer, for example for plants or organisms with tough cells. Optimized procedures for different sample types do exist; however a general procedure is not available (Görg et al 2000, Baudin & Bruneel, 2004Bruneel et al, 2005;Görg et al 2009). …”