Direct recovery of proteins into a free-liquid phase after preparative isoelectric focusing in immobilized pH gradients

Pier Giorgio Righetti, Antonio Morelli, Cecilia Gelfi, Reiner Westermeier

Research output: Contribution to journalArticlepeer-review

Abstract

A new method for electrophoretic retrieval of protein zones from Immobiline matrices is described, based on elution directly in a free liquid phase, rather than in ion-exchange beads or molecular sieves, as previously described. The chopped Immobiline gel is loaded on top of a 5% T stacking gel, 6-10 mm in height, and forced to transverse it and collect into a chamber, filled with 20% sucrose solution, closed on its anodic side by a dialysis sac. The trasfer is practically quantitative, for most proteins, after 30-60 min of zone electrophoresis at 10 W (300 V potential differential). Recovery of protein mass is in general better than 90%, while for enzyme activity is in the range of 60-80%. For preserving enzyme integrity, the following precautions are recommended: (1) short electrophoretic times; (2) avoidance of anodic oxidation; (3) chilling of the buffer in the anodic chamber; and (4) use of low levels (2-5 mM) of the specific enzyme substrate throughout the entire electrophoretic system (cathode, anode and gel plug).

Original languageEnglish
Pages (from-to)151-159
Number of pages9
JournalJournal of Biochemical and Biophysical Methods
Volume13
Issue number3
DOIs
Publication statusPublished - 1986

Keywords

  • immobilized pH gradients
  • isoelectric focusing
  • preparative electrophoresis

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics

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