ArticlePloS one2017
EDTA aggregates induce SYPRO orange-based fluorescence in thermal shift assay.
Article in PloS one, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 16 papers.
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Who cites it
16 citing papers in PubMed.
- Article
- The Photophysical Properties of the Protonation States of SYPROMolecules (Basel, Switzerland) · 2025Article
- Detection of an Intermediate in the Unfolding Process of the N-Terminal Domain of TDP-43.ACS omega · 2025Article
- Identification of Tunable, Environmentally Responsive Fluorogenic Dyes by High-Throughput Screening.ACS chemical biology · 2024Article
- Utilizing Thermal Shift Assay to Probe Substrate Binding to Selenoprotein O.Journal of visualized experiments : JoVE · 2024Article
- Fluorescence-Based Protein Stability Monitoring-A Review.International journal of molecular sciences · 2024Review
- Protocol for performing and optimizing differential scanning fluorimetry experiments.STAR protocols · 2023Article
- Thermal unfolding methods in drug discovery.Biophysics reviews · 2023Review
- An iron-chelating sulfonamide identified fromVirulence · 2022Article
- Biophysical and pharmacokinetic characterization of a small-molecule inhibitor of RUNX1/ETO tetramerization with anti-leukemic effects.Scientific reports · 2022Article
- Thermal Shift Assay for Small GTPase Stability Screening: Evaluation and Suitability.International journal of molecular sciences · 2022Article
- Biochemical, Enzymatic, and Computational Characterization of Recurrent Somatic Mutations of the Human Protein Tyrosine Phosphatase PTP1B in Primary Mediastinal B Cell Lymphoma.International journal of molecular sciences · 2022Article
- Structure of theCurrent research in structural biology · 2021Article
- In-depth interrogation of protein thermal unfolding data with MoltenProt.Protein science : a publication of the Protein Society · 2021Article
- Protein aggregation and immunogenicity of biotherapeutics.International journal of pharmaceutics · 2020Review
- nanoDSF:Frontiers in microbiology · 2020Article
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Authors and funding
10 authors.
Funding
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Abstract
Ethylenediaminetetraacetic acid (EDTA) is widely used in the life sciences as chelating ligand of metal ions. However, formation of supramolecular EDTA aggregates at pH > 8 has been reported, which may lead to artifactual assay results. When applied as a buffer component at pH ≈ 10 in differential scanning fluorimetry (TSA) using SYPRO Orange as fluorescent dye, we observed a sharp change in fluorescence intensity about 20°C lower than expected for the investigated protein. We hypothesized that this change results from SYPRO Orange/EDTA interactions. TSA experiments in the presence of SYPRO Orange using solutions that contain EDTA-Na+ but no protein were performed. The TSA experiments provide evidence that suggests that at pH > 9, EDTA4- interacts with SYPRO Orange in a temperature-dependent manner, leading to a fluorescence signal yielding a "denaturation temperature" of ~68°C. Titrating Ca2+ to SYPRO Orange and EDTA solutions quenched fluorescence. Ethylene glycol tetraacetic acid (EGTA) behaved similarly to EDTA. Analytical ultracentrifugation corroborated the formation of EDTA aggregates. Molecular dynamics simulations of free diffusion of EDTA-Na+ and SYPRO Orange of in total 27 μs suggested the first structural model of EDTA aggregates in which U-shaped EDTA4- arrange in an inverse bilayer-like manner, exposing ethylene moieties to the solvent, with which SYPRO Orange interacts. We conclude that EDTA aggregates induce a SYPRO Orange-based fluorescence in TSA. These results make it relevant to ascertain that future TSA results are not influenced by interference between EDTA, or EDTA-related molecules, and the fluorescent dye.
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