Home > Publications database > The Thermodynamic Fingerprints of Ultra-Tight Nanobody–Antigen Binding Probed via Two-Color Single-Molecule Coincidence Detection > print

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024 7 _ |a 10.3390/ijms242216379
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100 1 _ |a Schedler, Benno
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245 _ _ |a The Thermodynamic Fingerprints of Ultra-Tight Nanobody–Antigen Binding Probed via Two-Color Single-Molecule Coincidence Detection
260 _ _ |a Basel
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|b Molecular Diversity Preservation International
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520 _ _ |a Life on the molecular scale is based on a versatile interplay of biomolecules, a featurethat is relevant for the formation of macromolecular complexes. Fluorescence-based two-colorcoincidence detection is widely used to characterize molecular binding and was recently improvedby a brightness-gated version which gives more accurate results. We developed and establishedprotocols which make use of coincidence detection to quantify binding fractions between interactionpartners labeled with fluorescence dyes of different colors. Since the applied technique is intrinsicallyrelated to single-molecule detection, the concentration of diffusing molecules for confocal detectionis typically in the low picomolar regime. This makes the approach a powerful tool for determiningbi-molecular binding affinities, in terms of KD values, in this regime. We demonstrated the reliabilityof our approach by analyzing very strong nanobody-EGFP binding. By measuring the affinity atdifferent temperatures, we were able to determine the thermodynamic parameters of the bindinginteraction. The results show that the ultra-tight binding is dominated by entropic contributions.
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700 1 _ |a Yukhnovets, Olessya
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700 1 _ |a Lindner, Lennart
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700 1 _ |a Meyer, Alida
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700 1 _ |a Fitter, Jörg
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773 _ _ |a 10.3390/ijms242216379
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