Isoelectric focusing in immobilized pH gradients in the pH 10-11 range

Cecilia Gelfi, Maria Lusia Bossi, Bengt Bjellqvist, Pier Giorgio Righetti

Research output: Contribution to journalArticle

Abstract

With the synthesis of a new, strongly basic Immobiline (pK 10.3 at 10°C) it has been possible to formulate a new pH 10-11 recipe for focusing very alkaline proteins, not amenable to fractionation with conventional isoelectric focusing in carrier ampholyte buffers. In this formulation, water is added as an acidic Immobiline having pK = 14 and a unit molar concentration (or with a pK = 5 and standard 55.56 molarity) since around pH 11 its buffering power becomes significant. The gel contains a 'conductivity quencher', i.e. a density gradient incorporated in the matrix, with the dense region located on the cathodic side (pH 11) for (a) smoothing the voltage gradient on the separation cell and (b) reducing the anodic electrosmotic flow due to the next positive charge acquired by the matrix at pH 11 (1 mM excess protonated amino groups to act as counterions to the 1 mm OH- groups in the bulk water solution generated by the local value of pH 11). Excellent focusing is obtained for such alkaline proteins as lysozyme (pI 10.55), So-6 (a leaf protein, pI 10.49), cytochrome c (pI 10.45) and ribonuclease (pI 10.12).

Original languageEnglish
Pages (from-to)41-48
Number of pages8
JournalJournal of Biochemical and Biophysical Methods
Volume15
Issue number1
DOIs
Publication statusPublished - 1987

Keywords

  • Alkaline pH range
  • Immobilized pH gradien
  • Isoelectric focusing

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics

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