The Thermodynamic Fingerprints of Ultra-Tight Nanobody-Antigen Binding Probed via Two-Color Single-Molecule Coincidence Detection.
Van’t Hoff plot
antibody–antigen interaction
binding affinity
brightness-gated two-color coincidence detection (BTCCD)
burst analysis
confocal fluorescence microscopy
nanobodies
single-molecule fluorescence detection
thermodynamic parameters
Journal
International journal of molecular sciences
ISSN: 1422-0067
Titre abrégé: Int J Mol Sci
Pays: Switzerland
ID NLM: 101092791
Informations de publication
Date de publication:
15 Nov 2023
15 Nov 2023
Historique:
received:
11
10
2023
revised:
07
11
2023
accepted:
13
11
2023
medline:
27
11
2023
pubmed:
25
11
2023
entrez:
25
11
2023
Statut:
epublish
Résumé
Life on the molecular scale is based on a versatile interplay of biomolecules, a feature that is relevant for the formation of macromolecular complexes. Fluorescence-based two-color coincidence detection is widely used to characterize molecular binding and was recently improved by a brightness-gated version which gives more accurate results. We developed and established protocols which make use of coincidence detection to quantify binding fractions between interaction partners labeled with fluorescence dyes of different colors. Since the applied technique is intrinsically related to single-molecule detection, the concentration of diffusing molecules for confocal detection is typically in the low picomolar regime. This makes the approach a powerful tool for determining bi-molecular binding affinities, in terms of
Identifiants
pubmed: 38003569
pii: ijms242216379
doi: 10.3390/ijms242216379
pmc: PMC10671529
pii:
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
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