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Fl6500 spectrofluorometer

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The FL6500 spectrofluorometer is a laboratory instrument designed to measure the fluorescence properties of samples. It uses a light source, such as a xenon lamp, to excite the sample and detect the resulting fluorescence emission. The instrument is capable of performing wavelength scans, time-based measurements, and other fluorescence analysis techniques. The FL6500 provides accurate and reliable data to support various applications in research and analysis.

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Lab products found in correlation

Suprasil quartz cuvette, by Thermo Fisher Scientific (6 mentions) Spectrum fl software, by PerkinElmer (5 mentions) D fructose, by Merck Group (1 mentions)

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Spectrofluorometer

11 protocols using fl6500 spectrofluorometer

1

Competitive Binding Assay for SAL Proteins

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Affinity constants of ligands to the three SAL proteins were evaluated by competitive binding experiments, using 1-NPN as the fluorescent reporter. Spectra were recorded on a PerkinElmer FL 6500 spectrofluorometer in a right-angle configuration, at room temperature, with slits of 5 nm for both excitation and emission, using 1 cm path quartz cuvettes.
Binding of 1-NPN was measured by titrating a 2 μM solution of the protein in 50 mM Tris–HCl, pH 7.4 with aliquots of 1 mM methanol solution of 1-NPN to final concentrations of 2–16 μM. The excitation wavelength was set at 337 nm and intensities were recorded in correspondence with the peak maximum, around 408–412 nm, depending on the protein. Binding curves and dissociation constants for 1-NPN were obtained using Prism software.
Affinities of other ligands were evaluated in competitive binding experiments by treating a solution of the protein and 1-NPN in 50 mM Tris–HCl, pH 7.4, both at the concentration of 2 μM, with aliquots of 1 mM solutions in methanol of each chemical to final concentrations of 2–16 μM. Dissociation constants were calculated from the corresponding [IC]50 values (the concentration of each ligand halving the initial value of fluorescence), from the equation: Kd = [IC]50/1 + [1−NPN]/KNPN, where [1−NPN] is the concentration of free 1−NPN and KNPN the dissociation constant of the complex SAL/1-NPN.
Zaremska V., Fischer I.M., Renzone G., Arena S., Scaloni A., Knoll W, & Pelosi P. (2021). Reverse Chemical Ecology Suggests Putative Primate Pheromones. Molecular Biology and Evolution, 39(1), msab338.
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2

Glycation Sensitivity of Apolipoprotein A-I

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The glycation sensitivity was compared by incubating the purified lipid-free apoA-I (final 1 mg/mL) with 250 mM D-fructose (Sigma # F2793) in 200 mM potassium phosphate/0.02% sodium azide buffer (pH 7.4), as reported elsewhere [21 (link),37 (link)]. ApoA-I was incubated for up to 48 h in an atmosphere containing 5% CO2 at 37 °C. The extent of the advanced glycation reactions was determined by reading the fluorescence intensities at 370 nm (excitation) and 440 nm (emission), as described previously [56 (link)], using an FL6500 spectrofluorometer (Perkin-Elmer, Norwalk, CT, USA) with Spectrum FL software version 1.2.0.583 (Perkin–Elmer) and a 1 cm path-length Suprasil quartz cuvette (Fisher Scientific, Pittsburgh, PA, USA).
Cho K.H., Baek S.H., Nam H.S., Kim J.E., Kang D.J., Na H, & Zee S. (2023). Cuban Sugar Cane Wax Alcohol Exhibited Enhanced Antioxidant, Anti-Glycation and Anti-Inflammatory Activity in Reconstituted High-Density Lipoprotein (rHDL) with Improved Structural and Functional Correlations: Comparison of Various Policosanols. International Journal of Molecular Sciences, 24(4), 3186.
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3

Tryptophan Fluorescence Emission Spectra

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The primary sequence of EGF showed two Trp residues at the 49th and 50th amino acids in the C-terminal region. The Trp fluorescence was measured by determining the emission fluorescence maxima from the uncorrected spectra obtained on an FL6500 spectrofluorometer (Perkin-Elmer, Norwalk, CT, USA) using Spectrum FL software version 1.2.0.583 (Perkin-Elmer) using a 1 cm path-length Suprasil quartz cuvette (Fisher Scientific, Pittsburgh, PA, USA). The three equally diluted samples, i.e., Heberprot-P75®, Easyef®, and methyl-parabenzoic acid, were excited at 295 nm to avoid tyrosine fluorescence. The emission spectra were scanned from 190 to 900 nm at room temperature.
Cho K.H., Kim J.H., Nam H.S, & Kang D.J. (2022). Efficacy Comparison Study of Human Epidermal Growth Factor (EGF) between Heberprot-P® and Easyef® in Adult Zebrafish and Embryo under Presence or Absence Combination of Diabetic Condition and Hyperlipidemia to Mimic Elderly Patients. Geriatrics, 7(2), 45.
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4

Ligand Binding Experiments with EcorOBP15-m1

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Ligand binding experiments were performed
by using a PerkinElmer FL 6500 spectrofluorometer in a right-angle
configuration at room temperature and quartz cuvettes with a 1 cm
path. N-Phenyl-1-naphthylamine (1-NPN) was used as
a fluorescent probe at an excitation wavelength of 337 nm, and the
emission spectra were measured from 380 to 450 nm. In order to reach
concentrations of 2–16 μM, aliquots of a 1 mM methanol
solution of 1-NPN were added to a 2 μM solution of the protein
in 50 mM Tris–HCl buffer, pH 7.4. Intensity values were recorded
at a peak maximum at 421 nm for EcorOBP15-m1. Prism software was used
to calculate the dissociation constant of the complex protein/1-NPN
(https://www.graphpad.com/scientific-software/prism/). The affinity of ethyl hexanoate was evaluated by adding to a mixture
of the protein and 1-NPN at 2 μM concentration in 50 mM Tris–HCl
buffer (pH 7.4), aliquots of 1 mM methanol solutions of the ligand
to final concentration values of 2–16 μM. Dissociation
constants of the ligands were calculated from the corresponding [IC]50 values, using the equation where [IC]50 is the concentration
of each ligand halving the initial value of fluorescence, [1 –
NPN] is the concentration of free 1 – NPN, and K1–NPN is the dissociation constant of the complex
protein/(1 – NPN). The data can be found in Figure S3.
Kleinheinz D., D’Onofrio C., Carraher C., Bozdogan A., Ramach U., Schuster B., Geiß M., Valtiner M., Knoll W, & Andersson J. (2023). Activity of Single Insect Olfactory Receptors Triggered by Airborne Compounds Recorded in Self-Assembled Tethered Lipid Bilayer Nanoarchitectures. ACS Applied Materials & Interfaces, 15(40), 46655-46667.
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5

Fluorescent Ligand Binding Assay for OBPs

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Direct binding of fluorescent probes was monitored by adding aliquots of 1 mM methanol solution of the probe to a 2 μM solution of the protein in 50 mM Tris–HCl, pH 7.4 to final concentrations of 2 to 16 μM. The excitation wavelength was 337 nm and intensities were measured on the maximum of the peak, usually between around 410 and 420 nm. For VdesOBP1 and VdesOBP2, binding of ligands was evaluated by monitoring the intrinsic tryptophan quenching of the protein. Excitation wavelength was 295 nm and intensities were recorded around 335–340 nm. For VdesOBP4 and VdesOBP5, competitive binding experiments were performed by titrating a solution of protein and 1-NPN both at 2 μM in Tris–HCl buffer, pH 7.4 with 1 mM methanolic solutions of ligands to final concentrations of 2–16 μM. All measurements were performed with a FL 6500 spectrofluorometer (PerkinElmer); slits were set at 5 nm for both excitation and emission, and 1 cm path quartz cuvettes were used.
Dissociation constants for 1-NPN were evaluated using Prism software. Affinities of other ligands were calculated from the corresponding [IC]50 values (the concentration of each ligand halving the initial value of fluorescence), from the equation: Kd=IC50/1+1-NPN/KNPN where [1-NPN] is the concentration of free 1-NPN and KNPN is the dissociation constant of the complex OBP/1-NPN.
Amigues B., Zhu J., Gaubert A., Arena S., Renzone G., Leone P., Fischer I.M., Paulsen H., Knoll W., Scaloni A., Roussel A., Cambillau C, & Pelosi P. (2021). A new non-classical fold of varroa odorant-binding proteins reveals a wide open internal cavity. Scientific Reports, 11, 13172.
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6

Tryptophan Fluorescence in HDL3

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The change in the secondary structure upon treatment with OSO was observed at the wavelengths of maximum fluorescence (WMF) of the tryptophan residues in HDL3. The WMF was determined from the uncorrected spectra obtained on an FL6500 spectrofluorometer (Perkin-Elmer, Norwalk, CT, USA) using Spectrum FL software version 1.2.0.583 (Perkin-Elmer) using a 1 cm path-length Suprasil quartz cuvette (Fisher Scientific, Pittsburgh, PA, USA). The samples were excited at 295 nm to avoid tyrosine fluorescence. The emission spectra were scanned from 305 to 400 nm at room temperature.
Cho K.H., Kang D.J., Nam H.S., Kim J.H., Kim S.Y., Lee J.O, & Kim B.J. (2021). Ozonated Sunflower Oil Exerted Protective Effect for Embryo and Cell Survival via Potent Reduction Power and Antioxidant Activity in HDL with Strong Antimicrobial Activity. Antioxidants, 10(11), 1651.
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7

Tryptophan Fluorescence in HDL3 with Ferrous Ions

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The change in secondary structure upon treatment with ferrous ions was observed at the WMF of the tryptophan residues in HDL3. The WMF was determined from the uncorrected spectra obtained on an FL6500 spectrofluorometer (Perkin–Elmer, Norwalk, CT, USA) using Spectrum FL software version 1.2.0.583 (Perkin–Elmer) and a 1 cm path-length Suprasil quartz cuvette (Fisher Scientific, Pittsburgh, PA, USA). The samples were excited at 295 nm to avoid tyrosine fluorescence. As described previously, the emission spectra were scanned from 305 to 400 nm at room temperature [56 (link)].
Cho K.H., Nam H.S., Kim J.E., Na H.J., del Carmen Dominguez-Horta M, & Martinez-Donato G. (2023). CIGB-258 Exerts Potent Anti-Inflammatory Activity against Carboxymethyllysine-Induced Acute Inflammation in Hyperlipidemic Zebrafish via the Protection of Apolipoprotein A-I. International Journal of Molecular Sciences, 24(8), 7044.
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8

Tryptophan Fluorescence in HDL3

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The change in the secondary structure upon treatment with ferrous ion was observed at the wavelengths of maximum fluorescence (WMF) of the tryptophan residues in HDL3. The WMF was determined from the uncorrected spectra obtained on an FL6500 spectrofluorometer (Perkin-Elmer, Norwalk, CT, USA) using Spectrum FL software version 1.2.0.583 (Perkin-Elmer) and a 1 cm path-length Suprasil quartz cuvette (Fisher Scientific, Pittsburgh, PA, USA). The samples were excited at 295 nm to avoid tyrosine fluorescence. The emission spectra were scanned from 305 to 400 nm at room temperature.
Cho K.H., Kim J.E., Nam H.S., Kang D.J, & Na H.J. (2022). Anti-Inflammatory Activity of CIGB-258 against Acute Toxicity of Carboxymethyllysine in Paralyzed Zebrafish via Enhancement of High-Density Lipoproteins Stability and Functionality. International Journal of Molecular Sciences, 23(17), 10130.
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9

Fluorescence-based Binding Assay for BmorPBP1

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Dissociation constants were measured in solution along with the widely adopted fluorescence binding assay [9 (link),14 (link)]. Spectra were recorded on a PerkinElmer FL 6500 spectrofluorometer at room temperature in a right-angle configuration, with a 1-cm light path quartz cuvette and 5-nm slits for both excitation and emission.
First, the binding of BmorPBP1 to the fluorescent probe N-phenyl-1-naphthylamine (1-NPN) was measured by titrating a 2-μM solution of the protein in 50 mM Tris-Cl buffer at pH 7.4 with aliquots of 1 mM solution of 1-NPN in methanol to final concentrations of 2–16 μM. Excitation wavelength was 337 nm and intensity was recorded at the maximum of the emission peak, 408 nm. The affinities of other ligands were evaluated in competitive binding assays by titrating a solution of the protein and 1-NPN, both at the concentration of 2 μM, with aliquots of 1-mM methanol solutions of each chemical to final concentrations of 2–16 μM. The affinity to 1-NPN was calculated using Prism software. Dissociation constants of competing ligands were evaluated from the corresponding [IC]50 values (the concentration of each ligand halving the initial value of fluorescence), using the equation: Kd = [IC]50/1 + [1-NPN]/KNPN, where [1-NPN] is the concentration of free 1-NPN and KNPN the dissociation constant of the complex Protein/1-NPN.
Bonazza C., Zhu J., Hasler R., Mastrogiacomo R., Pelosi P, & Knoll W. (2021). Responses of the Pheromone-Binding Protein of the Silk Moth Bombyx mori on a Graphene Biosensor Match Binding Constants in Solution. Sensors (Basel, Switzerland), 21(2), 499.
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10

Fluorescence-Based Ligand Binding Assay

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Affinity constants were evaluated by the competitive fluorescent method, using a PerkinElmer FL 6500 spectrofluorometer in a right-angle configuration at room temperature and quartz cuvettes with a 1 cm path. The fluorescent probe N-phenyl-1-naphthylamine (1-NPN) was excited at 337 nm, and emission spectra were recorded between 380 and 450 nm. Binding of 1-NPN was evaluated by adding aliquots of a 1 mM methanol solution to reach final concentrations of 2–16 μM, to a 2 μM solution of the protein in 50 mM Tris-HCl buffer, pH 7.4. Intensity values were recorded at the peak maximum, around 412 nm. The dissociation constant of the complex protein/1-NPN was calculated using Prism software (https://www.graphpad.com/scientific-software/prism/, accessed on 10 February 2021). The affinities of other ligands were evaluated in competitive binding assays by adding aliquots of 1 mM methanol solutions of each ligand to final concentration values of 2 to 16 μM, to a mixture of the protein and 1-NPN, both at the concentration of 2 μM in 50 mM Tris-HCl buffer, pH 7.4. Dissociation constants of ligands were calculated from the corresponding [IC]50 values (the concentration of each ligand halving the initial value of fluorescence), using the equation:
where [1-NPN] is the concentration of free 1-NPN, and K1-NPN the dissociation constant of the complex protein/1-NPN.
D’Onofrio C., Knoll W, & Pelosi P. (2021). Aphid Odorant-Binding Protein 9 Is Narrowly Tuned to Linear Alcohols and Aldehydes of Sixteen Carbon Atoms. Insects, 12(8), 741.
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