Isoelectric protein purification by orthogonally coupled hydraulic and electric transports in a segmented immobilized pH gradient

Michel Faupel, Barbara Barzaghi, Cecilia Gelfi, Pier Giorgio Righetti

Research output: Contribution to journalArticlepeer-review

Abstract

A new method is described for preparative purification, based on isoelectric focusing on immobilized pH gradients. The principle is entirely new, as it is based on keeping the protein of interest isoelectric, in a flow-chamber, and focusing the impurities in the Immobiline gel. For this, a hydraulic flow is coupled orthogonally to an electric flow, sweeping away the non-isoelectric impurities from the recycling chamber. The sample flow-chamber is built in the centre of the apparatus, and is coupled to an upper and lower segment of an immobilized pH gradient. The protein to be purified is kept isoelectric in the flow-chamber and prevented from leaving it by arranging for the extremities of the immobilized pH gradient, forming the ceiling and the floor of this chamber, to have isoelectric points just higher (e.g. +0.05 pH units, on the cathodic side) and just lower (e.g. -0.05 pH units, on the anodic side) than the known pI of the species of interest. Macromolecules and small ions leave the flow chamber at a rate corresponding to a first order reaction kinetics (the plot of log C vs. time being linear). In general, for macromolecules, 12 h of recycling under current allow removal of 95% impurities. After 24 h of recycling, the protein of interest is more than 99.5% pure. The recoveries are very high (approaching 100%) as the sample under purification nerver enters the Immobiline gel and thus does not have extracted from a hydrophilic matrix, as typical of preparative gel electrophoresis.

Original languageEnglish
Pages (from-to)147-161
Number of pages15
JournalJournal of Biochemical and Biophysical Methods
Volume15
Issue number3-4
DOIs
Publication statusPublished - 1987

Keywords

  • Immobilized pH gradient
  • Isoelectric focusing
  • Preparative electrophoresis
  • Recycling apparatus

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics

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