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001-es BibID:BIBFORM072316
035-os BibID:(WoS)000425895600022 (Scopus)85041571964
Első szerző:Nagyné Szabó Ágnes Timea (vegyész)
Cím:The Effect of Fluorophore Conjugation on Antibody Affinity and the Photophysical Properties of Dyes / Szabó Ágnes, Szendi-Szatmári Tímea, Ujlaky-Nagy László, Rádi Ildikó, Vereb György, Szöllősi János, Nagy Peter
Dátum:2018
ISSN:0006-3495
Megjegyzések:Because the degree of labeling (DOL) of cell-bound antibodies, often required in quantitative fluorescence measurements, is largely unknown, we investigated the effect of labeling with two different fluorophores (AlexaFluor546, AlexaFluor647) in a systematic way using antibody stock solutions with different DOLs. Here, we show that the mean DOL of the cell-bound antibody fraction is lower than that of the stock using single molecule fluorescence measurements. The effect is so pronounced that the mean DOL levels off at approximately two fluorophores/IgG for some antibodies. We developed a method for comparing the average DOL of antibody stocks to that of the isolated, cell-bound fraction based on fluorescence anisotropy measurements confirming the aforementioned conclusions. We created a model in which individual antibody species with different DOLs, present in an antibody stock solution, were assumed to have distinct affinities and quantum yields. The model calculations confirmed that a calibration curve constructed from the anisotropy of antibody stocks can be used for determining the DOL of the bound fraction. The fluorescence intensity of the cell-bound antibody fractions and of the antibody stocks exhibited distinctly different dependence on the DOL. The behavior of the two dyes was systematically different in this respect. Fitting of the model to these data revealed that labeling with each dye affects quantum yield and antibody affinity differentially. These measurements also implied that fluorophores in multiply labeled antibodies exhibit self-quenching and lead to decreased antibody affinity, conclusions directly confirmed by steady-state intensity measurements and competitive binding assays. Although the fluorescence lifetime of antibodies labeled with multiple fluorophores decreased, the magnitude of this change was not sufficient to account for self-quenching indicating that both dynamic and static quenching processes occur involving H-aggregate formation. Our results reveal multiple effects of fluorophore conjugation, which must not be overlooked in quantitative cell biological measurements.
Tárgyszavak:Természettudományok Biológiai tudományok idegen nyelvű folyóiratközlemény külföldi lapban
folyóiratcikk
Megjelenés:Biophysical Journal. - 114 : 3 (2018), p. 688-700. -
További szerzők:Szendi-Szatmári Tímea (1989-) (molekuláris biológus) Ujlaky-Nagy László (1977-) (biofizikus) Rádi Ildikó Vereb György (1965-) (biofizikus, orvos) Szöllősi János (1953-) (biofizikus) Nagy Péter (1971-) (biofizikus)
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001-es BibID:BIBFORM077483
Első szerző:Ujlaky-Nagy László (biofizikus)
Cím:Flow cytometric FRET analysis of protein interactions / László Ujlaky-Nagy, Péter Nagy, János Szöllősi, György Vereb
Dátum:2018
Megjegyzések:In the past decades, investigation of protein?protein interactions in situ in living or intact cells has gained expanding importance as structure/function relationships proposed from bulk biochemistry and molecular modeling experiments required con?rmation at the cellular level. Fo ? rster (?uorescence) resonance energy transfer (FRET)-based methods are excellent tools for determining proximity and supramolecular organization of biomolecules at the cell surface or inside the cell. This could well be the basis for the increasing popularity of FRET. In fact, the number of publications exploiting FRET has exploded since the turn of the millennium. Interestingly, most applications are microscope-based, and only a fraction employs ?ow cytometry, even though the latter offers great statistical power owed to the potentially huge number of individually measured cells. However, with the increased availability of multi-laser ?ow cytometers, strategies to obtain absolute FRET ef?ciencies can now be relatively facilely implemented. In this chapter, we intend to provide generally useable protocols for measuring FRET in ?ow cytometry. After a concise theoretical introduction, recipes are provided for successful labeling techniques and measurement approaches. The simple, quenching-based population-level measurement, the classic ratiometric, intensity-based technique providing cell-by-cell actual FRET ef?ciencies, and a more advanced version of the latter, allowing for cell-by-cell auto?uorescence correction are described. An Excel macro pre-loaded with spectral data of the most commonly used ?uorophores is also provided for easy calculation of average FRET ef?ciencies. Finally, points of caution are given to help design proper experiments and critically interpret the results.
Tárgyszavak:Orvostudományok Elméleti orvostudományok könyvfejezet
Förster resonance energy transfer
Fluorescence resonance energy transfer
Flow cytometry
Protein interactions
Molecular proximity
FCET
Megjelenés:Flow Cytometry Protocols / eds. Teresa S. Hawley, Robert G. Hawley. - p. 393-419. -
További szerzők:Nagy Péter (1971-) (biofizikus) Szöllősi János (1953-) (biofizikus) Vereb György (1965-) (biofizikus, orvos)
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DOI
Intézményi repozitóriumban (DEA) tárolt változat
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