Immobilized pH gradients for isoelectric focusing. III. Preparative separations in highly diluted gels

Pier Giorgio Righetti, Cecilia Gelfi

Research output: Contribution to journalArticlepeer-review

Abstract

A further improvement on the preparative aspects of immobilized pH gradients (IPG) (J. Biochem. Biophys. Methods (1983) 8, 135-172) is described, based on the use of soft (highly diluted) polyacrylamide gels. While in conventional IPGs in 5%T gels an upper load limit of 40-45 mg protein/ml gel volume is found, in 2.5%T gels, containing the same amount of Immobiline, as much as 90 mg protein/ml gel can be applied, without overloading effects. This is an extraordinary amount of material to ba carried by a gel phase, and renders IPG by far the leading technique in any electrophoretic fractionation. A new, two-step casting technique, based on the formation of a %T step and a pH plateau around the application trench, is described. A new method for electrophoretic protein recovery from IPG gel strips, based on embedding on low-gelling agarose (37°C), is reported. The physico-chemical properties of highly diluted gels, in relation to their protein loading ability, are evaluated and discussed. It is recommended that diluted gels (e.g. 3.5%T) be used also in analytical runs, since sharper protein zones are obtained, due to the increased charge density on the polymer coil.

Original languageEnglish
Pages (from-to)103-119
Number of pages17
JournalJournal of Biochemical and Biophysical Methods
Volume9
Issue number2
DOIs
Publication statusPublished - 1984

Keywords

  • immobilized pH gradients
  • isoelectric focusing
  • preparative electrophoresis

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics

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