Post-capillary reaction detection in capillary electrophoresis based on the streptavidin-biotin interaction - Optimization and application to single cell analysis

Agatha Feltus, Nathaniel G. Hentz, Sylvia Daunert

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

A class-selective post-capillary reaction detection method for capillary electrophoresis is described in which a streptavidin-fluorescein isothiocyanate (streptavidin-FITC) conjugate is used to detect biotin moieties. The selective binding of biotin moieties to the streptavidin-FITC conjugate causes an enhancement of fluorescence proportional to the concentration of biotin present. After capillary electrophoresis the separated analytes react with streptavidin-FITC in a coaxial reactor and are then detected either by a benchtop spectrofluorometer (2.5 μM detection limit) or by an epi-fluorescence microscope (1·10-7 M detection limit). The method is used to examine biotinylated species in a crude mammalian cell lysate which was found to contain 83±3 fmol in 3600 cell volumes. In addition, it is used to examine the uptake of biotin by individual sea urchin oocytes. The results indicate that, in the oocytes, biocytin is the prevalent form of biotin and its concentration varies widely between cells (mean=2±2 μM).

Original languageEnglish (US)
Pages (from-to)381-392
Number of pages12
JournalJournal of Chromatography A
Volume918
Issue number2
DOIs
StatePublished - May 25 2001
Externally publishedYes

Keywords

  • Arbacia punctulata
  • Biotin
  • Derivatization, electrophoresis
  • Detection, electrophoresis

ASJC Scopus subject areas

  • Analytical Chemistry

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