Guanidine hydrochloride-induced unfolding was performed on 10 µM hIL-12 in the presence and absence of heparin. The equilibrium unfolding data was plotted to derive the melting concentration (Cm) and ΔG (H2O). The surface accessible non-polar surfaces in hIL-12, in the presence and absence of heparin, was monitored by 8-anilinonaphthalene-1-sulfonic acid (ANS) fluorescence. All fluorescence experiments were performed at 25 °C on a Hitachi F2500 spectrofluorimeter. The excitation wavelength was set to 280 nm and bandwidths for excitation and emission were set to 2.5 and 10 nm, respectively. A stock solution of 20 mM ANS solution was used for titration into 10 µM hIL12 in 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM sodium chloride. Samples were excited at 380 nm and emission spectra were monitored between 450 to 600 nm with a peak observed at 500 nm. Data from the ANS binding assay were overlaid to identify differences in surface hydrophobicity.
F 2500 spectrofluorimeter
Manufactured by Hitachi
Sourced in Japan
The F-2500 spectrofluorimeter is a laboratory instrument designed for fluorescence analysis. It is capable of measuring the fluorescence intensity of samples across a range of wavelengths. The core function of the F-2500 is to provide accurate and reliable fluorescence data for various applications.
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16 protocols using f 2500 spectrofluorimeter
1
Heparin Modulation of hIL-12 Stability
2
Fluorescence-based Tyrosine Kinetics Assay
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Tyrosine (2 mM) in 50 mM phosphate buffer, pH 8.0, was added with an equal amount of 1.63 mM ABTS● solution. Fluorescence spectra were measured immediately and at 10 min intervals in a Hitachi F-2500 spectrofluorimeter (Tokyo, Japan) at an excitation wavelength of 320 nm [9 (link),10 (link)], using excitation and emission slits of 5 nm.
3
Fluorescence Spectroscopy of Proteins
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Fluorescence measurements were performed on an F-2500 spectrofluorimeter (Hitachi, Japan) with a 150-W Xenon lamp, a 1.0-cm quartz cell and a thermostat bath. The width of both the excitation slit and emission slit was set at 5.0 nm. The operation software automatically corrected the spectral scan for the photomultiplier characteristics. Furthermore, fluorescence intensities were corrected for inner filter and dilution effects before any data analysis was carried out. The excitation was set at 280 and 295 nm, and the emission was collected in the range of 300–600 nm (excitation of Trp and Tyr).
4
Quantifying Glyphosate Retention in Plants
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The methodology adapted by González-Torralva et al. (2010) (link) was employed. Na-fluorescein was used as labeling reagent for determination of herbicide solution amount was retained. Seven plants from each population, in a completely random design, were treated with a solution containing 360 g ae ha-1 of glyphosate (0.5 of field rate) + 100 mg L-1 Na-fluorescein. When the solution applied on the plant’s foliage dried (20–25 min after application), the treated plants were cut off at ground level and washed with 50 mL of NaOH 5 mM in a test tube shaking it vigorously for 30 s. The washing solution was recovered in glass flasks and the absorbance of fluorescein was immediately measured at 490exc/510em nm (Hitachi F-2500 spectrofluorimeter). The plants were wrapped in filter paper envelopes and dried in a stove at 60°C for 1 week, and weighed. The retention was expressed in μL of glyphosate solution g-1 dry matter.
5
Intracellular Calcium Measurement Protocol
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Free cytosolic Ca2+ was measured using the ester Indo-AM, by procedures used previously in our lab [20] . Cells were incubated with 4 µM of Indo-AM dye in HBSS Hank's Balanced Salt Solution) for 1 h. Probenecid and pluronic acid were added at final concentrations of 0.03% and 2.7 mM respectively. Cells were washed twice after incubation with the dye and then incubated in HBSS for another hour to allow de-esterification. Subsequently, ACLAR sheets were placed in a quartz cuvette containing 1 ml of Ca2+ buffer at an inclination of approximately 45°. Ca2+ measurements were performed with an Hitachi F-2500 spectrofluorimeter at excitation wavelengths of 405 and 485 nm and the emission wavelength of 510 nm. In the post-incubation Ca2+ assay, cells were moved to new media by removing the strip containing the cells from one cuvette to another; the interval between media was less than 15 s. Following intracellular Ca2+ measurements, the ACLAR strip was subjected to treatment with 0.1% Triton X100 and 20 mM of EGTA in order to calculate maximum and minimum background calcium. Maximum calcium fluorescence (Fmax) was measured after Triton X100 treatment, and minimum calcium fluorescence (Fmin) after EGTA addition. Standard radiometric analysis using the Grynkewicz equation [21] (link) was conducted as in our prior studies [22] .
6
Morphological and Spectroscopic Analysis of Blends
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Morphological analysis of the blends was evaluated by Sirion XL30 FEG SEM. UV-Vis absorption spectra were recorded by a Shimadzu UV-1800 spectrophotometer in the 200–800 nm region. A Hitachi F-2500 spectrofluorimeter was used for fluorescence intensity measurements, where the band pass was 2.5 nm, the photomultiplier tube voltage was 400 V, and the scan speed was 5 nm s−1. We optimized the excitation point and excited A, AC, and AA at 370 nm, 380 nm, and 390 nm, respectively. Fluorescence lifetimes were measured by a time-correlated single-photon counting (TCSPC) Horiba DeltaFlex instrument. A 375 nm nano-LED was used as a light source for the experiment, where the pulse repetitions were set to 1 MHz. The IBH software was used for the decay data analysis, and the χ2 value is in the range between 0.98 to 1.1. An Alpha-N Analyser, (Novocontrol) was utilized for the electrical conductivity measurements at room temperature. The electromagnetic shielding efficiency was assessed by VNA (Anritsu Shockline) and associated waveguide with 8–12.4 GHz and 12–18 GHz frequency ranges.
7
Lipoplex Emission Spectroscopy Analysis
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Emission spectra of the lipoplex solutions were performed to quantify the interaction between DNA and liposomes. Measurements were carried out in a Hitachi F-2500 spectrofluorimeter interfaced to a PC for the recording and handling of the spectra and connected to a flow Lauda thermostat to maintain the temperature at 298.0 ± 0.1 K. A standard fluorescence quartz cell of 10 mm path length was used. Emission intensities of the lipoplexes prepared (DOPE and Ru(II)-based liposomes + DNA) were measured at different L/D and α values. DNA concentration used was 8.1 × 10−5 mol dm−3. The excitation and emission wavelengths used were 456 nm and 600 nm, respectively.
8
Foliar Herbicide Retention Assay
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Foliar herbicide retention assays were performed following the method adapted by Jiménez et al.27 (link). Plants were treated with a solution containing 40 g ai ha−1 of imazamox + 1.25 L ha−1 of adjuvant Dash (34.5% w/v methyl oleate/methyl palmitate) + 100 mg L−1 Na-fluorescein in the same treatment chamber used in dose-response assays. Na-fluorescein was used as a labeling reagent to determine the amount of herbicide solution retained. Once the herbicide solution from the leaf (20–25 min) was dry, the plants were cut at ground level and washed individually in Erlenmeyer’s containing 50 mL of NaOH 5 mM shaking them vigorously for 30 seconds. The washing solution was recovered in glass flasks and the fluorescein absorbance was immediately measured at 490exc/510em nm (Hitachi F-2500 spectrofluorimeter). The cut tissues were packed in cellulose envelopes and dried in an oven at 80 °C for 72 h. Ten plants of each cultivar were used in a completely random design. Retention was expressed as µL of imazamox solution per g of dry matter.
9
Intracellular Calcium Measurement with Fura-2
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Intracellular calcium was measured as previously described [61 (link)] using Fura-2 AM (1 μM final concentration) loaded cell suspensions and a Hitachi F2500 spectrofluorimeter.
10
Inhibitory Activity Measurement of Cysteine Peptidases
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