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Protocol to detect nucleotide-protein interaction in vitro using a non-radioactive competitive electrophoretic mobility shift assay
- Source :
- STAR Protocols, Vol 3, Iss 4, Pp 101730- (2022)
- Publication Year :
- 2022
- Publisher :
- Elsevier, 2022.
-
Abstract
- Summary: Electrophoretic mobility shift assay (EMSA) is a classical and popular approach for DNA/RNA protein-binding affinity detection in vitro. This protocol describes a competitive EMSA assay using digoxigenin (DIG)-labeled probe, which solves the safety issues and limitations attributed to the short lifespan of the 32P-radiolabeled DNA probe. We detail steps for DNA probe preparation, protein-DNA mixture coincubation, EMSA, and competitive EMSA process. We optimize the standard DIG-ddUTP-labeling EMSA protocol to high sensitivity with reproducible results.For complete details on the use and execution of this protocol, please refer to Feng et al. (2022). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Details
- Language :
- English
- ISSN :
- 26661667
- Volume :
- 3
- Issue :
- 4
- Database :
- Directory of Open Access Journals
- Journal :
- STAR Protocols
- Publication Type :
- Academic Journal
- Accession number :
- edsdoj.7b36a8ca63ee4e7c89f04eaa42c78736
- Document Type :
- article
- Full Text :
- https://doi.org/10.1016/j.xpro.2022.101730