Document Detail


Post-capillary reaction detection in capillary electrophoresis based on the streptavidin-biotin interaction. Optimization and application to single cell analysis.
MedLine Citation:
PMID:  11407585     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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).
Authors:
A Feltus; N G Hentz; S Daunert
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Journal of chromatography. A     Volume:  918     ISSN:  0021-9673     ISO Abbreviation:  J Chromatogr A     Publication Date:  2001 May 
Date Detail:
Created Date:  2001-06-15     Completed Date:  2001-12-04     Revised Date:  2009-01-15    
Medline Journal Info:
Nlm Unique ID:  9318488     Medline TA:  J Chromatogr A     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  381-92     Citation Subset:  IM    
Affiliation:
Department of Pharmaceutical Sciences, University of Kentucky, Lexington 40506-0055, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Biotin / chemistry*,  metabolism
Cells, Cultured
Electrophoresis, Capillary / methods*
Fluorescein-5-isothiocyanate
Hydrogen-Ion Concentration
Oocytes / metabolism
Rats
Sea Urchins
Spectrometry, Fluorescence
Streptavidin / chemistry*
Chemical
Reg. No./Substance:
3326-32-7/Fluorescein-5-isothiocyanate; 58-85-5/Biotin; 9013-20-1/Streptavidin

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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