ArticlePloS one2013
Analysis of high affinity self-association by fluorescence optical sedimentation velocity analytical ultracentrifugation of labeled proteins: opportunities and limitations.
Article in PloS one, 2013. 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, 33 citations in OpenAlex.
- Analysis of Giant-Shell CdSe/CdS Quantum Dots via Analytical Ultracentrifugation Combined with Spectrally Resolved Photoluminescence.Small methods · 2026Article
- On the utility of microfluidic systems to study protein interactions: advantages, challenges, and applications.European biophysics journal : EBJ · 2023Review
- Label-free methods for opticalPhysical chemistry chemical physics : PCCP · 2021Review
- Energetic and structural features of SARS-CoV-2 N-protein co-assemblies with nucleic acids.bioRxiv : the preprint server for biology · 2021Article
- Quantitative Analysis of Protein Self-Association by Sedimentation Velocity.Current protocols in protein science · 2020Article
- The structure of the extracellular domains of human interleukin 11α receptor reveals mechanisms of cytokine engagement.The Journal of biological chemistry · 2020Article
- Identification of the periplasmic DNA receptor for natural transformation of Helicobacter pylori.Nature communications · 2019Article
- Nucleic acid-induced dimerization of HIV-1 Gag protein.The Journal of biological chemistry · 2019Article
- Structural characterization of the RH1-LZI tandem of JIP3/4 highlights RH1 domains as a cytoskeletal motor-binding motif.Scientific reports · 2019Article
- All tubulins are not alike: Heterodimer dissociation differs among different biological sources.The Journal of biological chemistry · 2019Article
- Structural basis of transcriptional regulation by the HigA antitoxin.Molecular microbiology · 2019Article
- Ligand-induced conformational switch in an artificial bidomain protein scaffold.Scientific reports · 2019Article
- Sedimentation velocity analytical ultracentrifugation for characterization of therapeutic antibodies.Biophysical reviews · 2018Review
- The Challenge of Interpreting Glutamate-Receptor Ion-Channel Structures.Biophysical journal · 2017Review
- Article
- Use of fluorescence-detected sedimentation velocity to study high-affinity protein interactions.Nature protocols · 2017Article
- Article
- Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles.The EMBO journal · 2016Article
- On the acquisition and analysis of microscale thermophoresis data.Analytical biochemistry · 2016Article
- 3D-Printing for Analytical Ultracentrifugation.PloS one · 2016Article
Corrections and comments
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Authors and funding
5 authors at 3 institutions in 4 countries.
Funding
Abstract
Sedimentation velocity analytical ultracentrifugation (SV) is a powerful first-principle technique for the study of protein interactions, and allows a rigorous characterization of binding stoichiometry and affinities. A recently introduced commercial fluorescence optical detection system (FDS) permits analysis of high-affinity interactions by SV. However, for most proteins the attachment of an extrinsic fluorophore is an essential prerequisite for analysis by FDS-SV. Using the glutamate receptor GluA2 amino terminal domain as a model system for high-affinity homo-dimerization, we demonstrate how the experimental design and choice of fluorescent label can impact both the observed binding constants as well as the derived hydrodynamic parameter estimates for the monomer and dimer species. Specifically, FAM (5,6-carboxyfluorescein) was found to create different populations of artificially high-affinity and low-affinity dimers, as indicated by both FDS-SV and the kinetics of dimer dissociation studied using a bench-top fluorescence spectrometer and Förster Resonance Energy Transfer. By contrast, Dylight488 labeled GluA2, as well as GluA2 expressed as an EGFP fusion protein, yielded results consistent with estimates for unlabeled GluA2. Our study suggests considerations for the choice of labeling strategies, and highlights experimental designs that exploit specific opportunities of FDS-SV for improving the reliability of the binding isotherm analysis of interacting systems.
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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.