Casting immobilized pH gradients into cylindrical polyacrylamide gels

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

Research output: Contribution to journalArticle

Abstract

A novel method is described for casting immobilized pH gradients in polyacrylamide gel rods of small diameter (2 mm), based on the principle of rotational centrifugation. The tubes are filled vertically with equal volumes of dense and light solution (250 μl each) titrated to the extremes of the desired pH gradient, and then tilted at 2.5° to the level. After 5 min at rest, to allow for sliding of the two menisci to equilibrium position, the glass tubes are rotated for 3 min at 180 rpm, folowed by an additional 3 min at 180 rpm by reversing the sense of rotation. A homogeneous linear gradient is thus produced. The rotating platform is then raised to 90° and the gels allowed to polymerize under standard conditions. Formation of linear and reproducible pH gradinet is ensured by using stabilizing densisity gradients of low viscosity (0-5% glycerol, having a maximal ratio viscosity/density of 1.1).

Original languageEnglish
Pages (from-to)171-183
Number of pages13
JournalJournal of Biochemical and Biophysical Methods
Volume16
Issue number2-3
DOIs
Publication statusPublished - 1988

Keywords

  • Centrifugation by rotation
  • Immobilized pH gradient
  • Isoelectric focusing

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics

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