ArticleJournal of visualized experiments : JoVE2014
Electrophoretic mobility shift assay (EMSA) for the study of RNA-protein interactions: the IRE/IRP example.
Article in Journal of visualized experiments : JoVE, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 24 papers.
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Who cites it
24 citing papers in PubMed, 36 citations in OpenAlex.
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- CRP and HNF1A collaborate to regulate the progression of laryngeal cancer through the Wnt signaling pathway.Functional & integrative genomics · 2025Article
- Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens using a dual fluorescence readthrough reporter.Nucleic acids research · 2024Article
- Using Biotinylated Iron-Responsive Element to Analyze the Activity of Iron Regulatory Proteins.International journal of molecular sciences · 2024Article
- SHMT2 Mediates Small-Molecule-Induced Alleviation of Alzheimer Pathology Via the 5'UTR-dependent ADAM10 Translation Initiation.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2024Article
- Intracellular growth of Brucella is mediated by Dps-dependent activation of ferritinophagy.EMBO reports · 2023Article
- Optimization of Biotinylated RNA or DNA Pull-Down Assays for Detection of Binding Proteins: Examples of IRP1, IRP2, HuR, AUF1, and Nrf2.International journal of molecular sciences · 2023Article
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- RNA-controlled nucleocytoplasmic shuttling of mRNA decay factors regulates mRNA synthesis and a novel mRNA decay pathway.Nature communications · 2022Article
- The mutual interaction of glycolytic enzymes and RNA in post-transcriptional regulation.RNA (New York, N.Y.) · 2022Review
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- The risk variant rs11836367 contributes to breast cancer onset and metastasis by attenuating Wnt signaling via regulatingScience advances · 2022Article
- Next-generation sequencing: A new avenue to understand viral RNA-protein interactions.The Journal of biological chemistry · 2022Review
- Essential role of systemic iron mobilization and redistribution for adaptive thermogenesis through HIF2-α/hepcidin axis.Proceedings of the National Academy of Sciences of the United States of America · 2021Article
- Functional significance of gain-of-function H19 lncRNA in skeletal muscle differentiation and anti-obesity effects.Genome medicine · 2021Article
- Review
- Footprinting SHAPE-eCLIP Reveals Transcriptome-wide Hydrogen Bonds at RNA-Protein Interfaces.Molecular cell · 2020Article
- Ultra-fast conductive media for RNA electrophoretic mobility shift assays.BioTechniques · 2020Article
- Article
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Authors and funding
3 authors at 2 institutions in 1 country.
Funding
Abstract
RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron regulatory proteins, IRP1 and IRP2. They bind to iron responsive elements (IREs) within the untranslated regions (UTRs) of several target mRNAs, thereby controlling the mRNAs translation or stability. IRE/IRP interactions have been widely studied by EMSA. Here, we describe the EMSA protocol for analyzing the IRE-binding activity of IRP1 and IRP2, which can be generalized to assess the activity of other RNA-binding proteins as well. A crude protein lysate containing an RNA-binding protein, or a purified preparation of this protein, is incubated with an excess of(32) P-labeled RNA probe, allowing for complex formation. Heparin is added to preclude non-specific protein to probe binding. Subsequently, the mixture is analyzed by non-denaturing electrophoresis on a polyacrylamide gel. The free probe migrates fast, while the RNA/protein complex exhibits retarded mobility; hence, the procedure is also called "gel retardation" or "bandshift" assay. After completion of the electrophoresis, the gel is dried and RNA/protein complexes, as well as free probe, are detected by autoradiography. The overall goal of the protocol is to detect and quantify IRE/IRP and other RNA/protein interactions. Moreover, EMSA can also be used to determine specificity, binding affinity, and stoichiometry of the RNA/protein interaction under investigation.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.