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

被引：4

作者：

Wang, Fang ^{[2
]}

Yao, Ting ^{[1
]}

Yang, Wen ^{[1
]}

Wu, Pan ^{[1
]}

Liu, Yutao ^{[1
]}

Yang, Bin ^{[1
]}

机构：

[1] Nankai Univ, TEDA Inst Biol Sci & Biotechnol, TEDA, Tianjin 300457, Peoples R China

[2] Shenzhen Univ, Shenzhen Peoples Hosp 2, Shenzhen Inst Translat Med, Hlth Sci Ctr,Affiliated Hosp 1,Intens Care Unit, Shenzhen 518035, Peoples R China

来源：

STAR PROTOCOLS | 2022年 / 3卷 / 04期

基金：

中国国家自然科学基金;

关键词：

PROBES; EMSA;

D O I：

10.1016/j.xpro.2022.101730

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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-radio -labeled 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 reproduc-ible results. For complete details on the use and execution of this protocol, please refer to Feng et al. (2022).

引用

页数：20

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