2-DE with IPGs

• Published in: Electrophoresis Vol. 30; no. S1; pp. S122 - S132
• Main Authors: Görg, Angelika, Drews, Oliver, Lück, Carsten, Weiland, Florian, Weiss, Walter
• Format: Journal Article
• Language: English
• Published: Weinheim Wiley-VCH Verlag 01.06.2009; WILEY-VCH Verlag; WILEY‐VCH Verlag
• Subjects: 2-DE; Animals; Bacterial Proteins - analysis; Bacterial Proteins - chemistry; Electrophoresis, Gel, Two-Dimensional - history; Electrophoresis, Gel, Two-Dimensional - instrumentation; Electrophoresis, Gel, Two-Dimensional - methods; Electrophoresis, Gel, Two-Dimensional - standards; Equipment Design; History, 20th Century; History, 21st Century; Humans; Hydrogen-Ion Concentration; IPG; IPG-Dalt; Proteins - analysis; Proteins - chemistry; Proteomics - methods
• ISSN: 0173-0835; 1522-2683
• DOI: 10.1002/elps.200900051

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.