Improve Research Reproducibility A Bio-protocol resource
Concise Method
tooltip

Electrophoretic mobility shift assay (EMSA)

JY Junjun Yan
FL Feiqing Liu
ZG Zeyuan Guan
XY Xuhui Yan
XJ Xiaohuan Jin
QW Qiang Wang
ZW Zican Wang
JY Junjie Yan
DZ Delin Zhang
ZL Zhu Liu
SW Shan Wu
PY Ping Yin
request Request a Protocol
ask Ask a question
Favorite

For EMSA, the 27-nt ssDNA oligonucleotides (5′-AACTTCTGTCATTACATTAAGCTTTA A-3′) labeled at the 5′ end with FAM were synthesized by General Biosystems (Anhui) Co. Ltd. and annealed with an equimolar amount of the complementary strand as described above. An aliquot of 500 nM FAM-labeled dsDNA was mixed with increasing concentrations of wild-type or mutant complexes (0–15 μM) in 20 μL buffer containing 25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 20 mM DTT, 0.2 mg/mL BSA, 2 μg/mL Heparin, 50% glycerol and incubated on ice for 20 min. The obtained products were then resolved on 8% native acrylamide gels (37.5:1 acrylamide:bis-acrylamide) in 0.5× Tris-glycine buffer under an electric field of 10 V/cm for 2 h. Gels were visualized using Amersham Typhoon.

Copyright and License information: The Author(s) ©2023
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A