使用非放射性竞争电泳迁移率变动分析检测体外核苷酸-蛋白质相互作用的方案。

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

机构信息

Intensive Care Unit, Shenzhen Institute of Translational Medicine, Health Science Center, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen 518035, China.

TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, Tianjin 300457, China.

出版信息

STAR Protoc. 2022 Dec 16;3(4):101730. doi: 10.1016/j.xpro.2022.101730. Epub 2022 Sep 30.

DOI:10.1016/j.xpro.2022.101730

PMID:36181685

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9530670/

Abstract

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 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).

摘要

电泳迁移率变动分析（EMSA）是一种经典且流行的体外检测 DNA/RNA 与蛋白质结合亲和力的方法。本方案描述了一种使用地高辛（DIG）标记探针的竞争性 EMSA 检测法，该方法解决了 P-放射性标记 DNA 探针寿命短所带来的安全问题和局限性。我们详细介绍了 DNA 探针制备、蛋白-DNA 混合物共孵育、EMSA 和竞争性 EMSA 步骤。我们优化了标准的 DIG-ddUTP 标记 EMSA 方案，以实现高灵敏度和可重复的结果。如需了解本方案的详细使用和执行方法，请参考 Feng 等人（2022 年）。