To measure the tryptophan (Trp) fluorescence emission of ELIC, WT or W206F ELIC was diluted to 3 ml (0.02 mg/ml) and an emission spectrum was collected in a quartz fluorescence cell using a Fluoromax Plus Spectrofluorometer (Horiba Scientific). Excitation was set at 295 nm and an emission spectrum was collected from 320 to 380 nm. Trp fluorescence measurements with rapid mixing of agonist was carried out using the SX20 stopped-flow spectrofluorometer, outfitted with a 295 nm LED excitation source. Purified ELIC was diluted to 0.1 mg/ml in Trp Stopped-flow Buffer A (10 mM Tris pH 7.5, 100 mM NaCl, 0.05% DDM). A single mixing experiment was performed by mixing ELIC with equal volumes of Buffer A + propylamine. Propylamine was prepared at varying concentrations at 2x the final concentration. The instrument dead time was ~1 ms, enabling capture of most of the time course of change in Trp fluorescence. The time course of decrease in Trp fluorescence after rapid mixing with propylamine was fit to a single exponential to obtain a time constant for the rate of decrease and a measure of the extent of decrease. All replicates were taken from distinct samples.
Fluoromax plus spectrofluorometer
Manufactured by Horiba
Sourced in United States
The FluoroMax Plus spectrofluorometer is a laboratory instrument designed for the analysis of fluorescent samples. It is capable of performing excitation and emission scans, and can measure fluorescence intensity across a range of wavelengths.
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Lab products found in correlation
Vertex 80 hyperion2000, by Bruker (1 mentions)
Fluoromax 4p fluorescence spectrophotometer, by Horiba (1 mentions)
X ray powder diffractometer, by Bruker (1 mentions)
Axis supra, by Shimadzu (1 mentions)
Duetta, by Horiba (1 mentions)
Ht7800, by Hitachi (1 mentions)
Irtracer 100, by Shimadzu (1 mentions)
8 protocols using fluoromax plus spectrofluorometer
1
Tryptophan Fluorescence Emission Assay for ELIC
2
Cryogenic Photoluminescence Spectroscopy
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Low-temperature luminescence
spectra were recorded using a FluoroMax Plus spectrofluorometer (Horiba
Scientific) equipped with a cylindrical quartz dewar filled with liquid
nitrogen (at 77 K). 500 μL samples were transferred to quartz
tubes with 4 mm inner diameter and freeze-quenched into liquid nitrogen
before their introduction in the dewar. Emission spectra were obtained
with excitation at 310 nm.
spectra were recorded using a FluoroMax Plus spectrofluorometer (Horiba
Scientific) equipped with a cylindrical quartz dewar filled with liquid
nitrogen (at 77 K). 500 μL samples were transferred to quartz
tubes with 4 mm inner diameter and freeze-quenched into liquid nitrogen
before their introduction in the dewar. Emission spectra were obtained
with excitation at 310 nm.
3
Time-correlated Single-Photon Counting Analysis
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Time-correlated
single-photon
counting was conducted on a FluoroMax Plus spectrofluorometer (Horiba;
Irvine CA) equipped with a DeltaDiode Laser (DD-375L; Horiba) at 371
nm center wavelength using DeltaTime TCSPC. At least 10,000 counts
were recorded for all lifetime measurements measured at the maximum
fluorescence wavelength of roughly 590 nm. The samples were allowed
to incubate for several weeks at 20 °C before they were measured.
single-photon
counting was conducted on a FluoroMax Plus spectrofluorometer (Horiba;
Irvine CA) equipped with a DeltaDiode Laser (DD-375L; Horiba) at 371
nm center wavelength using DeltaTime TCSPC. At least 10,000 counts
were recorded for all lifetime measurements measured at the maximum
fluorescence wavelength of roughly 590 nm. The samples were allowed
to incubate for several weeks at 20 °C before they were measured.
4
Low-Temperature Luminescence Spectroscopy
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Low-temperature luminescence spectra were recorded using a FluoroMax Plus spectrofluorometer (Horiba Scientific) equipped with a cylindrical quartz Dewar filled with liquid nitrogen (at 77 K). A total of 500 µl samples were transferred to quartz tubes with 4 mm inner diameter and freeze-quenched into liquid nitrogen before their introduction in the Dewar.
5
Characterization of Fluorescent Gold Nanoclusters
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UV−vis absorption spectra were recorded using a UV-6000PC instrument, and the solution samples were prepared using ultrapure water as the solvent. All fluorescence spectra were obtained using a HORIBA FluoroMax-4P fluorescence spectrophotometer. Transmission electron microscopy (TEM) and HRTEM images were obtained from a JEM-F200 microscope. X-ray photoelectron spectroscopy (XPS) measurements were performed on a ESCALAB 250 high-performance electron spectrometer with monochromated Al Kα radiation as the excitation source. The binding energies were corrected by referencing the binding energies of C(1s) arising from the added hydrocarbons as an internal standard. Fourier transform infrared spectroscopy (FT-IR) was recorded using a Bruker Vertex80 + Hyperion2000 apparatus. The gold content in the clusters was measured using Inductively coupled plasma mass spectrometry (ICP-MS) on an Agilent 7800 instrument. The fluorescence quantum yield was measured using an Edinburgh-Steady State/Transient Fluorescence Spectrometer FLS1000. Photoluminescence lifetimes were measured by time-correlated single-photon counting (TCSPC) on a Horiba Fluoro max plus spectrofluorometer with a pulsed light-emitting diode (LED) (400 nm) as the excitation source.
6
Comprehensive Material Characterization
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UV-visible spectra and PL spectra were recorded using a Duetta, Horiba Scientific instrument at room temperature. The PL lifetime measurements were carried out using a FluoroMax Plus spectrofluorometer, Horiba Scientific at room temperature with excitation at 369 nm. XRD was performed on benchtop X-ray powder diffractometer (Bruker) using Cu-Kα radiation (λ = 0.154184 nm). Raman spectra were measured using a Horiba instrument at room temperature using the laser wavelength and spot size of 532 nm and ∼1 μm, respectively. XPS was measured by Axis Supra, Kratos under the base pressure of ∼2 × 10−9 Torr. Transmission electron microscopy (TEM) images were acquired using a Hitachi HT7800, operating at an accelerating voltage of 80 kV. FTIR spectroscopy was carried out using a Shimadzu IRTracer-100 at room temperature.
7
Fluorescence-based DNA-Resveratrol Binding
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Fluorescence experiments were performed using FluoroMax Plusspectrofluorometer(Horiba Scientific,USA). Fluorescence titrations were carried out in quartz cuvette with a fixed concentration of resveratrol at 10 µm. DNA solutionsweregradually addedin various D/Nratiosat room temperature. The emission spectra were recorded from 330 nm to 600 nm, with excitation wavelength at 318 nm. The excitation and emission slits were kept at 5 nm. Binding constant (Kb) and the number of binding sites (n) were calculated from the double logarithmic plot using the equation [50] (link).
WhereFo and F are the fluorescence intensities of the fluorophore in the absence and presence of different concentrations of DNA, respectively.
WhereFo and F are the fluorescence intensities of the fluorophore in the absence and presence of different concentrations of DNA, respectively.
8
Fluorescence-based DNA-Resveratrol Binding
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Fluorescence experiments were performed using FluoroMax Plusspectrofluorometer(Horiba Scientific,USA). Fluorescence titrations were carried out in quartz cuvette with a fixed concentration of resveratrol at 10 µm. DNA solutionsweregradually addedin various D/Nratiosat room temperature. The emission spectra were recorded from 330 nm to 600 nm, with excitation wavelength at 318 nm. The excitation and emission slits were kept at 5 nm. Binding constant (Kb) and the number of binding sites (n) were calculated from the double logarithmic plot using the equation [50] (link).
WhereFo and F are the fluorescence intensities of the fluorophore in the absence and presence of different concentrations of DNA, respectively.
WhereFo and F are the fluorescence intensities of the fluorophore in the absence and presence of different concentrations of DNA, respectively.
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