The Experts below are selected from a list of 10659 Experts worldwide ranked by ideXlab platform
James V. Staros - One of the best experts on this subject based on the ideXlab platform.
-
Preparation and characterization of Alexa Fluor 594-labeled epidermal growth factor for Fluorescence resonance energy transfer studies: application to the epidermal growth factor receptor.
Analytical biochemistry, 2004Co-Authors: Kristin B. Whitson, Joseph M. Beechem, Albert H. Beth, James V. StarosAbstract:We have prepared and characterized a new Fluorescent derivative of murine epidermal growth factor (EGF), Alexa Fluor 594-labeled EGF (A-EGF), for Fluorescence studies of EGF-EGF receptor interactions. We describe the synthesis of this derivative and its physical and biological characterization. The significant overlap between the excitation and the emission spectra of A-EGF makes this probe well suited to Fluorescence resonance energy homo-transfer. Using time-resolved Fluorescence to examine the oligomeric state of the EGF receptor, we have observed resonance energy homo-transfer of A-EGF bound to EGF receptors in cells, but not of A-EGF bound to EGF receptors in membrane vesicles. Our results, interpreted in the context of recent crystallographic studies of the ligand-binding domains of EGF receptors, suggest that observed Fluorescence resonance energy transfer does not result from transfer within receptor dimers, but rather results from transfer within higher-order oligomers. Furthermore, our results support a structural model for oligomerization of EGF receptors in which dimers are positioned head-to-head with respect to the ligand-binding site, consistent with the head-to-head interactions observed between adjacent receptor dimers by X-ray crystallography.
Joseph Beechem - One of the best experts on this subject based on the ideXlab platform.
-
Development of homogeneous binding assays based on Fluorescence resonance energy transfer between quantum dots and Alexa Fluor Fluorophores.
Analytical biochemistry, 2006Co-Authors: Theo T. Nikiforov, Joseph BeechemAbstract:We studied the Fluorescence resonance energy transfer (FRET) between quantum dots emitting at 565, 605, and 655 nm as energy donors and Alexa Fluor Fluorophores with absorbance maxima at 594, 633, 647, and 680 nm as energy acceptors. As a first step, we prepared covalent conjugates between all three types of quantum dots and each of the Alexa Fluor Fluorophores that could act as an energy acceptor. All of these conjugates displayed efficient resonance energy transfer. Then we prepared covalent conjugates of these quantum dots with biotin, Fluorescein, and cortisol and established that the binding of these conjugates to suitable Alexa Fluor-labeled antibodies and streptavidin (in the case of biotin) can be efficiently detected by measuring the resonance energy transfer in homogeneous solutions. Finally, based on these observations, competitive binding assays for these three small analytes were developed. The performance of these assays as a function of the degree of labeling of the quantum dots was evaluated. It was found that decreasing the degree of loading of the quantum dots leads to decreases of the limits of detection. The results show the great potential of this FRET system for the development of new homogeneous binding assays.
Thorben Cordes - One of the best experts on this subject based on the ideXlab platform.
-
molecular and spectroscopic characterization of green and red cyanine Fluorophores from the Alexa Fluor and af series
ChemPhysChem, 2021Co-Authors: Christian Gebhardt, Martin Lehmann, Maria M. Reif, Martin Zacharias, Gerd Gemmecker, Thorben CordesAbstract:The use of Fluorescence techniques has an enormous impact on various research fields including imaging, biochemical assays, DNA-sequencing and medical technologies. This has been facilitated by the development of numerous commercial dyes with optimized photophysical and chemical properties. Often, however, information about the chemical structures of dyes and the attached linkers used for bioconjugation remain a well-kept secret. This can lead to problems for research applications where knowledge of the dye structure is necessary to predict or understand (unwanted) dye-target interactions, or to establish structural models of the dye-target complex. Using a combination of optical spectroscopy, mass spectrometry, NMR spectroscopy and molecular dynamics simulations, we here investigate the molecular structures and spectroscopic properties of dyes from the Alexa Fluor (Alexa Fluor 555 and 647) and AF series (AF555, AF647, AFD647). Based on available data and published structures of the AF and Cy dyes, we propose a structure for Alexa Fluor 555 and refine that of AF555. We also resolve conflicting reports on the linker composition of Alexa Fluor 647 maleimide. We also conducted a comprehensive comparison between Alexa Fluor and AF dyes by continuous-wave absorption and emission spectroscopy, quantum yield determination, Fluorescence lifetime and anisotropy spectroscopy of free and protein-attached dyes. All these data support the idea that Alexa Fluor and AF dyes have a cyanine core and are a derivative of Cy3 and Cy5. In addition, we compared Alexa Fluor 555 and Alexa Fluor 647 to their structural homologs AF555 and AF(D)647 in single-molecule FRET applications. Both pairs showed excellent performance in solution-based smFRET experiments using alternating laser excitation. Minor differences in apparent dye-protein interactions were investigated by molecular dynamics simulations. Our findings clearly demonstrate that the AF-Fluorophores are an attractive alternative to Alexa- and Cy-dyes in smFRET studies or other Fluorescence applications.
-
Molecular and spectroscopic characterization of green and red cyanine Fluorophores from the Alexa Fluor and AF series
2020Co-Authors: Christian Gebhardt, Martin Lehmann, Maria M. Reif, Martin Zacharias, Thorben CordesAbstract:Abstract The use of Fluorescence techniques has had an enormous impact on various research fields including imaging, biochemical assays, DNA-sequencing and medical technologies. This has been facilitated by the availability of numerous commercial dyes, but often information about the chemical structures of dyes (and their linkers) are a well-kept secret. This can lead to problems for applications where a knowledge of the dye structure is necessary to predict (unwanted) dye-target interactions, or to establish structural models of the dye-target complex. Using a combination of spectroscopy, mass spectrometry and molecular dynamics simulations, we here investigate the molecular structures and spectroscopic properties of dyes from the Alexa Fluor (Alexa Fluor 555 and 647) and AF series (AF555, AF647, AFD647). Based on available data and published structures of the AF and Cy dyes, we present two possible structures for Alexa Fluor 555. We also resolve conflicting reports on the linker composition of Alexa Fluor 647. A comprehensive comparison between Alexa Fluor and AF dyes by continuous-wave absorption and emission spectroscopy, quantum yield determination, Fluorescence lifetime and anisotropy spectroscopy of free and protein-attached dyes, supports the suggestion that the Alexa Fluor and AF dyes have a high degree of structural similarity. In addition, we compared Alexa Fluor 555 and Alexa Fluor 647 to their structural homologs AF555 and AF(D)647 in single-molecule FRET applications. Both pairs showed excellent performance in solution-based smFRET experiments using alternating laser excitation demonstrating that the AF-Fluorophores are an attractive alternative to Alexa- and Cy-dyes for smFRET studies, and suggesting their usefulness for other Fluorescence applications.
Kristin B. Whitson - One of the best experts on this subject based on the ideXlab platform.
-
Preparation and characterization of Alexa Fluor 594-labeled epidermal growth factor for Fluorescence resonance energy transfer studies: application to the epidermal growth factor receptor.
Analytical biochemistry, 2004Co-Authors: Kristin B. Whitson, Joseph M. Beechem, Albert H. Beth, James V. StarosAbstract:We have prepared and characterized a new Fluorescent derivative of murine epidermal growth factor (EGF), Alexa Fluor 594-labeled EGF (A-EGF), for Fluorescence studies of EGF-EGF receptor interactions. We describe the synthesis of this derivative and its physical and biological characterization. The significant overlap between the excitation and the emission spectra of A-EGF makes this probe well suited to Fluorescence resonance energy homo-transfer. Using time-resolved Fluorescence to examine the oligomeric state of the EGF receptor, we have observed resonance energy homo-transfer of A-EGF bound to EGF receptors in cells, but not of A-EGF bound to EGF receptors in membrane vesicles. Our results, interpreted in the context of recent crystallographic studies of the ligand-binding domains of EGF receptors, suggest that observed Fluorescence resonance energy transfer does not result from transfer within receptor dimers, but rather results from transfer within higher-order oligomers. Furthermore, our results support a structural model for oligomerization of EGF receptors in which dimers are positioned head-to-head with respect to the ligand-binding site, consistent with the head-to-head interactions observed between adjacent receptor dimers by X-ray crystallography.
Sanjeev R. Inamdar - One of the best experts on this subject based on the ideXlab platform.
-
Multidonor surface energy transfer from Alexa Fluor dyes to gold nanoparticles: a quest for innovative sensor applications
Journal of Nanophotonics, 2020Co-Authors: Mallikarjun K. Patil, M. G. Kotresh, Laxmi S. Inamdar, Sanjeev R. InamdarAbstract:We report energy transfer (ET) from two dyes: Alexa Fluor 514 (AF514) and Alexa Fluor 532 (AF532) to gold nanoparticles (AuNPs) of three different sizes (10, 30, and 53 nm) employing steady-state and time-resolved Fluorescence measurements. The results show that the Fluorescence intensity and Fluorescence lifetimes of donor (D) molecules AF514 and AF532 decrease with increase in the concentration of acceptor (A) AuNPs (2 to 10 μM) upon interaction with AuNPs, thereby confirming the occurrence of ET between D and A. This clearly suggests that these two Alexa Fluor molecules act as efficient donors and AuNPs as excellent acceptors. Interestingly, the Forster distance (Ro) determined for these dyes varies from 212 to 550 A with increasing size of AuNPs and suggests that the ET from AF514 and AF532 to AuNPs is essentially obeying surface energy transfer (SET) process following 1 / d4 distance dependence. As is well known, Forster resonance energy transfer is efficient for separation distances of up to 100 A, beyond which its efficiency decreases. Thus, the present results follow dipole-surface type ET from molecule dipole (AF514 and AF532) to nanometal (Au) surface. The influence of size and distance on the SET from AF514 and AF532 to AuNPs is discussed. Further, the quenching of donor Fluorescence in the presence of AuNPs and nonradiative ET are analyzed using Stern–Volmer plots. Our study is an experimental quest to explore the potential of such dye–noble metal NPs pairs performing as sensitive chemical and biosensors.
-
A combined solvatochromic shift and TDDFT study probing solute-solvent interactions of blue Fluorescent Alexa Fluor 350 dye: Evaluation of ground and excited state dipole moments.
Spectrochimica acta. Part A Molecular and biomolecular spectroscopy, 2019Co-Authors: Mallikarjun K. Patil, M. G. Kotresh, Sanjeev R. InamdarAbstract:Abstract Herein we report, the effect of solvents on absorption and Fluorescence spectra of Alexa Fluor-350 labelled Fluorescent dye examined both experimentally and computationally. The steady state absorption and Fluorescence measurements are carried out in a series of solvents to explore their solvatochromism and to determine its dipole moments. To this end, different empirical solvatochromic models like Bilot-Kawaski, Lippert-Mataga, Bakhshiev, Kawaski-Chamma-Viallet and Reichardt models are assessed against Alexa Fluor 350 dye to determine the singlet excited and ground state dipole moments. Computational studies were carried out to optimize ground and excited geometries using density functional theory (DFT) and time dependent density functional theory (TD-DFT), respectively, in vacuum. Additionally, this study encompasses estimation of the electronic transition energies from the ground to first excited state of dye employing TD-DFT. Further, TD-DFT has been combined with integral equation formalism of the polarizable continuum model (IEF-PCM) to calculate various solute-solvent interaction potentials which are then compared with experimental values. The highest occupied molecular orbital energy (HOMO), lowest unoccupied molecular orbital energy (LUMO), the energy gap, chemical hardness (η), softness (σ), electronegativity (χ) and chemical potential (μ) were estimated. Mulliken atomic charge, natural population analysis (NPA) and molecular electrostatic potential (MEP) map are correlated using density functional theory. The experimentally obtained ground and excited state dipole moments are compared with the ones obtained from computational and the results are discussed. NBO analysis is carried out to investigate the intramolecular charge transfer interactions and stabilization energy within the studied molecule.