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Electrophoretic Heterogeneity Limits the Utility of Streptavidin-β-Galactosidase as a Probe in Free Zone Capillary Electrophoresis Separations.
MedLine Citation:
PMID:  23325029     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
Single molecule assays were performed on streptavidin-β-galactosidase using a capillary electrophoresis-based protocol in order to assess the suitability of single molecule β-galactosidase assays for adaptation to the detection of single copies of target DNA. The conjugate was found to have a heterogeneous catalytic rate, showing an average rate of 44,000 ± 24,000 min(-1), which is similar to that of the unmodified enzyme. Electrophoretic mobility was also measured on individual molecules and determined to be -1.32 × 10(-4) ± 0.19 × 10(-4) cm(2)V(-1)s(-1). The variance in mobility was several times that reported for the unmodified enzyme. The electrophoretic heterogeneity was found to result in the formation of a broad window of peaks in the resultant electropherograms of free zone separations of small plugs of streptavidin-β-galactosidase. This range of mobilities largely overlapped with that of the conjugate bound to primer and plasmid containing a target DNA sequence. This overlap suggests that the separation of free conjugate from that bound to target DNA, which is a requirement for application of the single enzyme molecule assay to the detection of target DNA sequences, is not plausible using free zone capillary electrophoresis.
Authors:
Douglas B Craig; Anna Henderson
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2013-1-17
Journal Detail:
Title:  The protein journal     Volume:  -     ISSN:  1875-8355     ISO Abbreviation:  Protein J.     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2013-1-17     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101212092     Medline TA:  Protein J     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Affiliation:
Chemistry Department, University of Winnipeg, 599 Portage Avenue, Winnipeg, MB, Canada, d.craig@uwinnipeg.ca.
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