Preparative isoelectric focusing in immobilized pH gradients. II. A case report

Cecilia Gelfi, Pier Giorgio Righetti

Research output: Contribution to journalArticlepeer-review

Abstract

The preparative aspects of isoelectric focusing (IEF) in immobilized pH gradients have been investigated as a function of the following parameters: environmental ionic strength (I), gel geometry and shape of pH gradient. As model proteins, hemoglobin (Hb) A and a minor, glycosylated component (HbA1c), with a ΔpI = 0.04 pH units, have been selected. The load capacity increases almost linearly, as a function of progressively higher I values, from 0.5 × up to 2 × molarity of buffering Immobiline (pK 7.0) to abruptly reach a plateau at 3 × concentration of buffering ion. The load capacity also increases almost linearly as a function of gel thickness from 1 to 5 mm, without apparently levelling off. When decreasing the pH interval from 1 pH unit (pH 6.8-7.8) to 1 2 pH unit (pH 7.05-7.55) the amount of protein loaded in the HbA zone could be increased by 40%. In 5 mm thick gels, at 2 × pK 7.0 Immobiline concentration, over a 1 2 pH unit span, up to 350 mg HbA (in a 12.5 × 11 cm gel) could be loaded in a single zone, the load limit of the system being around 45 mg protein/ml gel volume.

Original languageEnglish
Pages (from-to)157-172
Number of pages16
JournalJournal of Biochemical and Biophysical Methods
Volume8
Issue number2
DOIs
Publication statusPublished - 1983

Keywords

  • immobilized pH gradient
  • isoelectric focusing
  • preparative electrophoresis

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics

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