ArticleAnalytical biochemistry2013
Determining the affinity and stoichiometry of interactions between unmodified proteins in solution using Biacore.
Article in Analytical biochemistry, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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Who cites it
13 citing papers in PubMed, 21 citations in OpenAlex.
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- TRIM21 and Fc-engineered antibodies: decoding its complex antibody binding mode with implications for viral neutralization.Frontiers in immunology · 2024Article
- Studying the Structures of Relaxed and Fuzzy Interactions: The Diverse World of S100 Complexes.Frontiers in molecular biosciences · 2021Review
- Antigen-binding affinity and thermostability of chimeric mouse-chicken IgY and mouse-human IgG antibodies with identical variable domains.Scientific reports · 2019Article
- Time-resolved neutron scattering provides new insight into protein substrate processing by a AAA+ unfoldase.Scientific reports · 2017Article
- Nanophotonic detection of freely interacting molecules on a single influenza virus.Scientific reports · 2015Article
- Synergistic Binding of Vascular Endothelial Growth Factor-A and Its Receptors to Heparin Selectively Modulates Complex Affinity.The Journal of biological chemistry · 2015Article
- Studying macromolecular complex stoichiometries by peptide-based mass spectrometry.Proteomics · 2015Review
- Production of functional human vitamin A transporter/RBP receptor (STRA6) for structure determination.PloS one · 2015Article
- Fab-based bispecific antibody formats with robust biophysical properties and biological activity.mAbs · 2015Article
- Analysis of protein interactions with picomolar binding affinity by fluorescence-detected sedimentation velocity.Analytical chemistry · 2014Article
- Mutation of nonessential cysteines shows that the NF-κB essential modulator forms a constitutive noncovalent dimer that binds IκB kinase-β with high affinity.Biochemistry · 2013Article
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Authors and funding
5 authors at 2 institutions in 1 country.
Funding
Abstract
We describe a general Biacore method for measuring equilibrium binding affinities and stoichiometries for interactions between unmodified proteins and their unmodified ligands free in solution. Mixtures of protein and ligand are preequilibrated at different ratios in solution and then analyzed by Biacore using a sensor chip surface that detects only unbound analyte. Performing the Biacore analysis under mass transport limited conditions allows the concentration of unbound analyte to be determined from the initial velocity of binding. Plots of initial velocity versus the concentration of the varied binding partner are fitted to a quadratic binding equation to give the affinity and stoichiometry of binding. We demonstrate the method using soluble Her2 extracellular domain binding to monovalent, bivalent, and trivalent forms of an anti-Her2 antibody. The affinity we measured agrees with that obtained from conventional Biacore kinetic analysis, and the stoichiometries for the resulting 1:1, 1:2, and 1:3 complexes were confirmed by gel filtration with in-line light scattering. The method is applicable over an affinity range of approximately 100 pM to 1 μM and is particularly useful when there is concern that covalently modifying one or the other binding partner might affect its binding properties or where multivalency might otherwise complicate a quantitative analysis of binding.
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