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| Content Provider | Springer Nature Link |
|---|---|
| Author | Craig, Douglas B. Henderson, Anna |
| Copyright Year | 2013 |
| Abstract | 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. |
| Starting Page | 81 |
| Ending Page | 88 |
| Page Count | 8 |
| File Format | |
| ISSN | 15723887 |
| Journal | The Protein Journal |
| Volume Number | 32 |
| Issue Number | 2 |
| e-ISSN | 15734943 |
| Language | English |
| Publisher | Springer US |
| Publisher Date | 2013-01-17 |
| Publisher Place | Boston |
| Access Restriction | One Nation One Subscription (ONOS) |
| Subject Keyword | Avidin conjugate Biotinylated primer Capillary electrophoresis Catalytic heterogeneity Electrophoretic mobility Electrophoretic heterogeneity Fluorescence detection β-galactosidase Separation Single molecule Streptavidin conjugate Target DNA Bioorganic Chemistry Biochemistry Organic Chemistry Animal Anatomy / Morphology / Histology |
| Content Type | Text |
| Resource Type | Article |
| Subject | Organic Chemistry Analytical Chemistry Biochemistry Bioengineering |
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