Casting immobilized pH gradients into cylindrical polyacrylamide gels

Bossi, Maria Luisa; Bossi, Oscar; Gelfi, Cecilia; Righetti, Pier Giorgio

doi:10.1016/0165-022x(88)90028-0

Cited by 4 publications

(2 citation statements)

References 19 publications

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“…1) 15, 30, which finally went into industrial production in 1991. Attempts by Righetti's 31 and Hochstrasser's 32 groups to run IPG IEF in capillaries or gel rods – complementary to carrier ampholyte 2‐DE in gel rods – were not successful. In contrast to those attempts, our approach was based on flat‐bed IPG gels.…”

Section: Milestonesmentioning

confidence: 99%

2‐DE with IPGs

et al. 2009

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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.

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Section: Milestonesmentioning

confidence: 99%

2‐DE with IPGs

et al. 2009

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“…The key development in improving reproducibility of 2-D electrophoresis was the introduction of immobilized pH gradients (IPG) that almost replaced carrier ampholyte-based pH gradient in tube gel (Gorg et al, 1988a;Gorg et al, 1988b). Although procedure for casting of IPG gels was described in details (Bossi et al, 1988;Gianazza, 1999), precast IPG gels with wide or various narrow range of pH are now commercially available (but more expensive). IPG gels with narrow pH gradients are most helpful to separate closely arrayed spots for accurate analysis of protein spots of interest.…”

Section: Low Reproducibilitymentioning

confidence: 99%

Two-Dimensional Polyacrylamide Gel Electrophoresis - A Practical Perspective

Magdeldin¹,

Zhang²,

Xu³

et al. 2012

Gel Electrophoresis - Principles and Basics

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On the computational approach to immobilized pH gradients

1991

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The unified treatment for computing the pH of complex mixtures of mono- and polyprotic buffers, including ampholytes, as utilized in the gradient simulation program PGS, is presented. Its ability to compute pH, buffering power and ionic strength is shown by discussing a few simulations. The problems arising in the automatic formulation of optimal mixtures are presented, as well as the merits and limits of several target functions utilized in such optimizations. It is shown that no universal target function exists and that a proper optimization method should account for the fact that more than one formulation is possible for a given pH range.

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Casting immobilized pH gradients into cylindrical polyacrylamide gels

Cited by 4 publications

References 19 publications

2‐DE with IPGs

2‐DE with IPGs

Two-Dimensional Polyacrylamide Gel Electrophoresis - A Practical Perspective

On the computational approach to immobilized pH gradients

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