Isoelectric focusing and two-dimensional electrophoresis of tubulin using immobilized pH gradients under denaturing conditions

Deborah J. Field, James C. Lee

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

Modifications of the LKB Immobiline isoelectric focusing (IEF) technique are described for use under conditions that solubilize and denature most proteins (8 m urea and 2% Nonidet-P40). This procedure permits pH gradients that are four- to fivefold shallower than previously available with conventional ampholine-IEF procedures. It can also be used as a first dimension in two-dimensional gel electrophoresis. The advantage of the stable ultranarrow pH gradient is demonstrated by directly comparing the resolution of vertebrate brain tubulins using (i) denaturing conventional ampholine-IEF and (ii) denaturing Immobiline-IEF. Analysis of tubulin on the Immobiline-IEF gel increases the separation distance between the individual tubulins and distinguishes differences among tubulin samples that could not be resolved by conventional ampholine isoelectric focusing.

Original languageEnglish (US)
Pages (from-to)584-592
Number of pages9
JournalAnalytical Biochemistry
Volume144
Issue number2
DOIs
StatePublished - Feb 1 1985

Keywords

  • denaturing gels
  • immobilized pH gradients
  • isoelectric focusing
  • tubulin heterogeneity
  • two-dimensional gels

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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