Post-capillary reaction detection in capillary electrophoresis based on the streptavidin-biotin interaction. Optimization and application to single cell analysis.
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 microM detection limit) or by an epi-fluorescence microscope (1 x 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 microM).
Feltus
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A
Department of Pharmaceutical Sciences, University of Kentucky, Lexington 40506-0055, USA.
Hentz
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NG
Daunert
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S
D000818:Animals; D001710:Biotin / Q000737:chemistry* / Q000378:metabolism; D002478:Cells, Cultured; D019075:Electrophoresis, Capillary / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D006863:Hydrogen-Ion Concentration; D009865:Oocytes / Q000378:metabolism; D051381:Rats; D012617:Sea Urchins; D013050:Spectrometry, Fluorescence; D019809:Streptavidin / Q000737:chemistry*
D001710:Biotin; D019809:Streptavidin; D016650:Fluorescein-5-isothiocyanate