2-DE with IPGs.
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.
Görg
Angelika
A
Proteomics Department, Technische Universität München, Freising, Germany. Angelika.Gorg@wzw.tum.de
Drews
Oliver
O
Lück
Carsten
C
Weiland
Florian
F
Weiss
Walter
W
D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000266:history / Q000295:instrumentation* / Q000379:methods* / Q000592:standards; D004867:Equipment Design; D049673:History, 20th Century; D049674:History, 21st Century; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011506:Proteins / Q000032:analysis / Q000737:chemistry; D040901:Proteomics / Q000379:methods*
D001426:Bacterial Proteins; D011506:Proteins