4. Conventional Isoelectric Focusing in Gel Matrices and Capillaries and Immobilized pH Gradients

  1. Mahmoud Hamdan1 and
  2. Pier Giorgio Righetti2

Published Online: 27 JAN 2005

DOI: 10.1002/0471709158.ch4

Proteomics Today: Protein Assessment and Biomarkers Using Mass Spectrometry, 2D Electrophoresis, and Microarray Technology

Proteomics Today: Protein Assessment and Biomarkers Using Mass Spectrometry, 2D Electrophoresis, and Microarray Technology

How to Cite

Hamdan, M. and Righetti, P. G. (2005) Conventional Isoelectric Focusing in Gel Matrices and Capillaries and Immobilized pH Gradients, in Proteomics Today: Protein Assessment and Biomarkers Using Mass Spectrometry, 2D Electrophoresis, and Microarray Technology, John Wiley & Sons, Inc., Hoboken, NJ, USA. doi: 10.1002/0471709158.ch4

Author Information

  1. 1

    GlaxoSmithKline Research Centre in Verona, Italy

  2. 2

    University of Verona, Italy

Publication History

  1. Published Online: 27 JAN 2005
  2. Published Print: 11 FEB 2005

ISBN Information

Print ISBN: 9780471648178

Online ISBN: 9780471709152

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Keywords:

  • Isoelectric focusing (IEF);
  • gel matricies;
  • capillaries;
  • immobilized pH gradients (IPG);
  • electrophoretic equipment;
  • polymerisation cassette;
  • polyacrylamide matrix;
  • reagents;
  • gel formulations;
  • carrier ampholytes;
  • gel preparation;
  • electrophoresis;
  • gel polymerisation;
  • protein staining;
  • Micellar Coomassie Blue G-250;
  • Coomassie Blue R-250/CuSo4;
  • Coomassie Blue R-250/sulphosalicylic acid;
  • Coomassie Blue G-250/urea/perchloric acid;
  • silver stain;
  • protein detection;
  • pH gradients;
  • capillary isoelectric focusing (cIEF)

Summary

After a brief historical survey, isoelectric focusing (IEF), on all of its possible variants, is here extensively dealt with. It is customary to start with conventional isoelectric focusing in amphoteric buffers, the soluble compounds described in the early 1960 and that laid the foundations of all IEF techniques. The chemistry of the carrier ampholyte buffers is extensively reviewed, together with the standard equipment for performing IEF in thin and ultrathin gel slabs. The chemistry of the polyacrylamide matrix is also discussed in depth, in that this is by far the most popular support not only for conventional IEF, but especially for its most advanced version of immobilized pH gradients (IPG). From that point of view, novel matrices coupling extreme resistance to hydrolysis with very high hydrophilicity are presented and evaluated. Here too a host of stains developed in purpose for IEF matrices are presented, including different variants of micellar Coomassie stains, of such a high sensitivity as to be dubbed “blue silver”. A number of silver stains, especially adapted to IEF matrices, are also presented. The section on conventional IEF ends by discussing all the problems connected with the soluble, amphoteric buffers, so that the transition to IPGs appears as its natural evolution. Immobilized pH gradients, in fact, seem to represent the ultimate in resolution and stability of any focusing technique, coupled to an extraordinary versatility in engineering the pH gradients, from ultranarrow to very extended, including non-linear gradients for analysis of whole tissue lysates. Since IPGs are today by far the preferred first separation dimension in two dimensional map analysis, particular care is taken in describing extended pH gradients, and in general rules for their generation and optimization. It would not be fair to end such an extensive chapter without mentioning capillary isoelectric focusing (cIEF), a unique approach to IEF techniques in that it is fully automated, offer on line detection and can be easily interfaced with mass spectrometry devices for identification of eluted peaks. The advantage of cIEF is that the separation is carried out in a free liquid phase, thus avoiding the lengthy and cumbersome procedures of extracting proteins from gel matrices, as in all other focusing techniques. An important spin-off of cIEF is zone electrophoresis in isoelectric, amphoteric buffers: by being essentially stationary, such buffers permit separations in the zone electrophoretic mode but with the very high voltage gradients typical of cIEF and of any other focusing technique.