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Title: Electrophoretic Mobility Shift Assays using Infrared-Fluorescent DNA Probes
Protein-DNA binding interactions are critical in several biological processes, especially the regulation of gene expression at the level of transcription initiation. An important technique for studying these interactions is the electrophoretic mobility shift assay (EMSA), whereby protein-DNA complexes are resolved on the basis of their mass:charge ratio using native polyacrylamide gel electrophoresis (nPAGE). Here we describe EMSA using PCR-generated, near infrared-fluorescent DNA probes, and IR fluorescence imaging to qualitatively and quantitatively study the interaction of transcriptional regulatory proteins from thermophilic organisms with different DNAs. Direct imaging of IR fluorophore-labeled DNA probes is advantageous because it provides high sensitivity (subnanomolar) without the need for intermediate staining steps or costly and problematic radiolabeled probes, thereby providing a more affordable and sensitive option to image protein-DNA on polyacrylamide gels by techniques such as EMSA.  more » « less
Award ID(s):
1714778
PAR ID:
10098102
Author(s) / Creator(s):
;
Date Published:
Journal Name:
Protocols.io
ISSN:
2473-1838
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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