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 Citations (Scopus)

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

Fingerprint

Single-Cell Analysis
Capillary electrophoresis
Streptavidin
Capillary Electrophoresis
Biotin
Fluorescein
Oocytes
Limit of Detection
Fluorescence
Sea Urchins
Cell Size
Microscopes
Cells
isothiocyanic acid

Keywords

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

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Post-capillary reaction detection in capillary electrophoresis based on the streptavidin-biotin interaction - Optimization and application to single cell analysis. / Feltus, Agatha; Hentz, Nathaniel G.; Daunert, Sylvia.

In: Journal of Chromatography A, Vol. 918, No. 2, 25.05.2001, p. 381-392.

Research output: Contribution to journalArticle

@article{c9f167cdb928459ba6e51812b1419183,
title = "Post-capillary reaction detection in capillary electrophoresis based on the streptavidin-biotin interaction - Optimization and application to single cell analysis",
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).",
keywords = "Arbacia punctulata, Biotin, Derivatization, electrophoresis, Detection, electrophoresis",
author = "Agatha Feltus and Hentz, {Nathaniel G.} and Sylvia Daunert",
year = "2001",
month = "5",
day = "25",
doi = "10.1016/S0021-9673(01)00742-7",
language = "English",
volume = "918",
pages = "381--392",
journal = "Journal of Chromatography",
issn = "0021-9673",
publisher = "Elsevier",
number = "2",

}

TY - JOUR

T1 - Post-capillary reaction detection in capillary electrophoresis based on the streptavidin-biotin interaction - Optimization and application to single cell analysis

AU - Feltus, Agatha

AU - Hentz, Nathaniel G.

AU - Daunert, Sylvia

PY - 2001/5/25

Y1 - 2001/5/25

N2 - 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).

AB - 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).

KW - Arbacia punctulata

KW - Biotin

KW - Derivatization, electrophoresis

KW - Detection, electrophoresis

UR - http://www.scopus.com/inward/record.url?scp=0035947443&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0035947443&partnerID=8YFLogxK

U2 - 10.1016/S0021-9673(01)00742-7

DO - 10.1016/S0021-9673(01)00742-7

M3 - Article

C2 - 11407585

AN - SCOPUS:0035947443

VL - 918

SP - 381

EP - 392

JO - Journal of Chromatography

JF - Journal of Chromatography

SN - 0021-9673

IS - 2

ER -