Protocol to detect nucleotide-protein interaction in vitro using a non-radioactive competitive electrophoretic mobility shift assay

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 life...

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Detalles Bibliográficos
Autores principales: Wang, Fang, Yao, Ting, Yang, Wen, Wu, Pan, Liu, Yutao, Yang, Bin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2022
Materias:
Protocol
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9530670/
https://www.ncbi.nlm.nih.gov/pubmed/36181685
http://dx.doi.org/10.1016/j.xpro.2022.101730
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author Wang, Fang
Yao, Ting
Yang, Wen
Wu, Pan
Liu, Yutao
Yang, Bin
author_facet Wang, Fang
Yao, Ting
Yang, Wen
Wu, Pan
Liu, Yutao
Yang, Bin
author_sort Wang, Fang
collection PubMed
description 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 (32)P-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).
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spelling pubmed-95306702022-10-05 Protocol to detect nucleotide-protein interaction in vitro using a non-radioactive competitive electrophoretic mobility shift assay Wang, Fang Yao, Ting Yang, Wen Wu, Pan Liu, Yutao Yang, Bin STAR Protoc Protocol 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 (32)P-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). Elsevier 2022-09-30 /pmc/articles/PMC9530670/ /pubmed/36181685 http://dx.doi.org/10.1016/j.xpro.2022.101730 Text en © 2022 The Authors https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Protocol
Wang, Fang
Yao, Ting
Yang, Wen
Wu, Pan
Liu, Yutao
Yang, Bin
Protocol to detect nucleotide-protein interaction in vitro using a non-radioactive competitive electrophoretic mobility shift assay
title Protocol to detect nucleotide-protein interaction in vitro using a non-radioactive competitive electrophoretic mobility shift assay
title_full Protocol to detect nucleotide-protein interaction in vitro using a non-radioactive competitive electrophoretic mobility shift assay
title_fullStr Protocol to detect nucleotide-protein interaction in vitro using a non-radioactive competitive electrophoretic mobility shift assay
title_full_unstemmed Protocol to detect nucleotide-protein interaction in vitro using a non-radioactive competitive electrophoretic mobility shift assay
title_short Protocol to detect nucleotide-protein interaction in vitro using a non-radioactive competitive electrophoretic mobility shift assay
title_sort protocol to detect nucleotide-protein interaction in vitro using a non-radioactive competitive electrophoretic mobility shift assay
topic Protocol
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9530670/
https://www.ncbi.nlm.nih.gov/pubmed/36181685
http://dx.doi.org/10.1016/j.xpro.2022.101730
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