Isoelectric focusing followed by electrophoresis of proteins for visualizing their titration curves by zymogram and immunofixation

E. Gianazza, C. Gelfi, G. Righetti

Research output: Contribution to journalArticle

Abstract

After isoelectric focusing followed by electrophoresis at right angles in the same gel slab, it is possible to visualize the titration curve of proteins by zymograms or immunofixation even of an unpurified sample. This information can be very useful for the selection of the proper purification strategy by charge-dependent methods, e.g. ion-exchange chromatography, zone and disc electrophoresis and isotachophoresis. The titration curve also gives information on the stability of the protein as a function of the prevailing pH of the medium, in the pH 3-10 range. A region of instability is found for most proteins in acidic conditions, below pH 4.5, while most proteins are stable in the alkaline pH region at least up to pH 10. The best method for developing zymograms and immunoprints appears to be the 'sandwich technique', by which a thin agarose slab, cast on an hydrophilic polyester sheet, and impregnated with appropriate reagents, is left in contact with a polyacrylamide gel thin layer used to generate the titration curves.

Original languageEnglish
Pages (from-to)65-75
Number of pages11
JournalJournal of Biochemical and Biophysical Methods
Volume3
Issue number2
DOIs
Publication statusPublished - 1980

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics

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