Soft immobilized pH gradient gels in proteome analysis

A follow-up

Maurizio Bruschi, Luca Musante, Giovanni Candiano, Gian Marco Ghiggeri, Ben Herbert, Francesca Antonucci, Pier Giorgio Righetti*

*Corresponding author for this work

Research output: Contribution to journalArticle

50 Citations (Scopus)

Abstract

As a follow-up of a previous work on two-dimensional map analysis utilizing soft (< 4%T) immobilized pH gradient (IPG) matrices in the first dimension (Candiano et al., Electrophoresis 2002, 23, 292-297), we have further optimized the preparation of such dilute IPG gels. One important step for obtaining an even reswelling of the entire IPG strip along the pH 3-10 interval is a washing step in 100 mM citric acid. It appears as though after rinsing off the excess acid in distilled water, a gradient of this tricarboxylic acid remains trapped into the IPG matrix, from almost nil at the acidic gel region to substantially higher amounts in its basic counterpart. This gradient helps in obtaining a uniform reswelling of the IPG strip, since carboxyl groups are more heavily hydrated than amino groups. The combined effects of uniform reswelling and of diluting the gel matrix favor penetration of large macromolecules (> 200 kDa) and allow for better spot resolution and for the display of a substantially higher number of spots also in the 30-60 000 Da region. A delipidation step in tri-n-butylphosphate:acetone:methanol (1:12:1) appears to substantially improve spot focusing and greatly diminish streaking and smearing of spots in all regions of the pH gradient.

Original languageEnglish
Pages (from-to)821-825
Number of pages5
JournalProteomics
Volume3
Issue number6
DOIs
Publication statusPublished - 1 Jun 2003

Keywords

  • Platelets
  • Porous immobilized pH gradient gels
  • Red blood cells
  • Two-dimensional maps

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    Bruschi, M., Musante, L., Candiano, G., Ghiggeri, G. M., Herbert, B., Antonucci, F., & Righetti, P. G. (2003). Soft immobilized pH gradient gels in proteome analysis: A follow-up. Proteomics, 3(6), 821-825. https://doi.org/10.1002/pmic.200300361