Electrophoretic mobility shift scanning using an automated infrared DNA sequencer
Electrophoretic mobility shift assay (EMSA) is widely used in the study of sequence-specific DNA-binding proteins, including transcription factors and mismatch binding proteins. We have established a non-radioisotope-based protocol for EMSA that features an automated DNA sequencer with an infrared f...
| Veröffentlicht in: | BioTechniques. - 1993. - 31(2001), 5 vom: 16. Nov., Seite 1056-8, 1060, 1062 |
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| Weitere Verfasser: | , , , |
| Format: | Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
2001
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| Zugriff auf das übergeordnete Werk: | BioTechniques |
| Schlagworte: | Journal Article DNA-Binding Proteins DNA 9007-49-2 |
| Zusammenfassung: | Electrophoretic mobility shift assay (EMSA) is widely used in the study of sequence-specific DNA-binding proteins, including transcription factors and mismatch binding proteins. We have established a non-radioisotope-based protocol for EMSA that features an automated DNA sequencer with an infrared fluorescent dye (IRDye) detection unit. Our modification of the elec- trophoresis unit, which includes cooling the gel plates with a reduced well-to-read length, has made it possible to detect shifted bands within 1 h. Further, we have developed a rapid ligation-based method for generating IRDye-labeled probes with an approximately 60% cost reduction. This method has the advantages of real-time scanning, stability of labeled probes, and better safety associated with nonradioactive methods of detection. Analysis of a promoter from an industrially important filamentous fungus, Aspergillus oryzae, in a prototype experiment revealed that the method we describe has potential for use in systematic scanning and identification of the functionally important elements to which cellular factors bind in a sequence-specific manner |
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| Beschreibung: | Date Completed 18.04.2002 Date Revised 28.09.2018 published: Print Citation Status MEDLINE |
| ISSN: | 0736-6205 |