Electrofocusing of integral membrane proteins in mixtures of zwitterionic and nonionic detergents

Leonard M Hjelmeland, Daniel W. Nebert, Andreas Chrambach

Research output: Contribution to journalArticle

49 Scopus citations

Abstract

In an attempt to fractionate mouse liver cytochrome P-450 in its native state, electrofocusing systems were examined under conditions in which the surface net charge of solubilized proteins was preserved. A mixture of the zwitterionic detergent, SB14, and the nonionic detergent, Triton X-100, appeared capable of completely solubilizing intergral membrane proteins. Since charge properties were not altered, it was possible, for the first time, to focus basic membrane proteins in such detergent mixtures. The pH gradients (pI range 7-11) formed in the presence of these detergents were sufficiently stable to allow electrofocusing to the steady state of the solubilized membrane proteins. By the criterion of patter constancy, these conditions were achieved within 15 h, 0-4°C, at 200 V in 6-cm gels of 5% T 15% CBis with 0.1 n H2SO4 and 0.1 n KOH as anolyte and catholyte, respectively. It was expected that the native state of solubilized proteins could be maintained in such systems. Cytochrome P-450 proved to be denatured, however, by concentrations of these detergents required for complete solubilization of mouse liver endoplasmic reticulum.

Original languageEnglish (US)
Pages (from-to)201-208
Number of pages8
JournalAnalytical Biochemistry
Volume95
Issue number1
DOIs
StatePublished - 1979
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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