Quasi-isoelectric buffers for protein analysis in a fast alternative to conventional capillary zone electrophoresis

Paolo Antonioli, Martha E. Mendieta, Roberto Sebastiano, Attilio Citterio, Gabriel Peltre, Jean Marc Busnel, Stephanie Descroix, Giovanni Candiano, Pier Giorgio Righetti

Research output: Contribution to journalArticlepeer-review

Abstract

Two different approaches are here reported for obtaining ultra-narrow pI cuts from 2-pH unit wide carrier ampholyte ranges, as commercially available, for use as quasi-isoelectric buffers in capillary electrophoresis separations of proteins. One of them uses multicompartment electrolyzers endowed with isoelectric membranes (Immobiline technology); the other employs the Rotofor equipment. Although the first approach results in more precise pI cuts, the latter technique is much faster, easier to handle and permits the immediate collection of 20 fractions in a single run. This results in ultra-narrow, ca. 0.1-pH unit intervals, uniformly spaced apart along the original wider gradient utilized for the fractionation. It is here shown that such quasi-isoelectric buffers, especially those in the pH 8-9 interval, have the unique property of coating the silica wall, thus preventing interaction of the proteins with the silica surface, that would otherwise totally disrupt the separation. On the contrary, such a shielding is not obtained in control, non isoelectric buffers (such as phosphate), that give very poor separations in uncoated capillaries. It is hypothesized that such a unique shielding effect is due to the oligo-amino backbone of the carrier ampholytes, typically composed (in the Vesterberg's synthetic approach) of 4-6 nitrogens spaced apart by ethylene moieties. Although such oligoprotic buffers should bear, in the isoelectric state, just one positive and one negative charge, they might be transiently ionized upon contact with the silanols, thus inducing a cooperative binding to the silica wall.

Original languageEnglish
Pages (from-to)19-25
Number of pages7
JournalJournal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Volume833
Issue number1
DOIs
Publication statusPublished - Mar 20 2006

Keywords

  • Fractionation techniques
  • Isoelectric buffer
  • Ultra-narrow-pH ampholytes cuts
  • Uncoated-capillary CZE

ASJC Scopus subject areas

  • Biochemistry

Fingerprint

Dive into the research topics of 'Quasi-isoelectric buffers for protein analysis in a fast alternative to conventional capillary zone electrophoresis'. Together they form a unique fingerprint.

Cite this