Purified HeRs were prepared in 150 mM NaCl, 50 mM Tris, and 0.02% DDM solution at pH 7.0. UV/VIS spectroscopy was used to measure the absorption spectra of the purified HeRs with the Shimadzu UV–visible spectrophotometer (UV-2450).
Uv 2450 uv visible spectrophotometer
Manufactured by Shimadzu
Sourced in Japan
The UV-2450 is a UV-visible spectrophotometer manufactured by Shimadzu. It is designed to measure the absorbance or transmittance of light in the ultraviolet and visible spectrum. The instrument can be used to quantify the concentration of various analytes in a sample by analyzing the absorption of light at specific wavelengths.
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Lab products found in correlation
Vario macro cube element analyzer, by Elementar (2 mentions)
Autoanalyzer 3, by SEAL Analytical (2 mentions)
Venusil xbp c18 column, by Agela Technologies (1 mentions)
Enspire multimode plate reader, by PerkinElmer (1 mentions)
Avance 3 300 mhz, by Bruker (1 mentions)
Mcp pmt, by Edinburgh Instruments (1 mentions)
Fourier transform infrared spectrometer, by PerkinElmer (1 mentions)
Zetasizer nano instrument, by Malvern Panalytical (1 mentions)
Infinite m200 multimode microplate reader, by Tecan (1 mentions)
Particle size analyzer, by Malvern Panalytical (1 mentions)
Mira3 feg sem, by TESCAN (1 mentions)
Jem 2010 transmission electron microscope, by JEOL (1 mentions)
Jem 2100f high resolution transmission electron microscope, by JEOL (1 mentions)
Paragon 1000 ftir spectrometer, by PerkinElmer (1 mentions)
21 protocols using uv 2450 uv visible spectrophotometer
1
Purification and Characterization of HeRs
2
HPLC Quantification of Phenylalanine
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The final concentrations of Phe in the supernatant were determined using HPLC (Agilent 1200, Frederick, CO, USA) and Venusil XBP C18 column (4.6 mm × 150 mm × 5 μm, 150 Å, Agela Technologies, Tianjin, China) analysis. During the measurement of Phe, the flow rate of the mobile phase at 90:10 (v/v) of methanol–water solution was 1.0 mL min−1.
The TW-80 and TX-100 were detected at 233 and 223 nm with an ultraviolet–visible (UV–visible) spectrophotometer (UV–Visible Spectrophotometer, UV-2450, SHIMADZU, Kyoto, Japan).
All experiments were performed in triplicate. The means and standard deviations were calculated by using SPSS 23.0.
The TW-80 and TX-100 were detected at 233 and 223 nm with an ultraviolet–visible (UV–visible) spectrophotometer (UV–Visible Spectrophotometer, UV-2450, SHIMADZU, Kyoto, Japan).
All experiments were performed in triplicate. The means and standard deviations were calculated by using SPSS 23.0.
3
Spectroscopic Analysis of Purified Retinal Proteins
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UV/Vis spectroscopy was used to measure the absorption spectra of the purified GRs by the Shimadzu UV-visible spectrophotometer (UV-2450) (Shimadzu, Japan). UV/Vis spectra and fluorescence spectra measurements were performed using purified GR wild-type and mutants in 150 mM NaCl, 50 mM Tris, and 0.02% DDM solution at pH 7.0. Excitation spectra for retinal fluorescence emissions were performed by EnSpire Multimode Plate Reader (PerkinElmer, USA); emission wavelength at 720 nm, and samples were prepared in 0.02% DDM solution at pH 4.0. The chromophore bleach solution included 3 M urea and 500 mM hydroxylamine, while the total chromophores were extracted by methanol. Triplicate experiments were conducted, and the optical density value was used to calculate a binding ratio. An extinction coefficient of 42,800 M−1 cm−1 for retinal57 (link) and 118,000 M−1 cm−1 for canthaxanthin58 was used, and then the value was used to determine protein level in fluorescence intensity and proton pumping assay since the maximum purification of protein from cell and chromophore extraction was achieved without a significant loss. Absorption spectra were baseline corrected with a defined linear fit as shown in Supplementary Fig. 2 , and the Origin 9.0 Program was used to perform the data fitting and calculations.
4
In Vitro Release and Functionality Evaluation of Sensor Molecules
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To evaluate the release of sensor molecules (CAM, DAF-FM DA, or MBs), nanosensors were dispersed in PBS at 37 °C. The absorbance of the supernatants at 495 nm was obtained at different time points using a UV-2450 UV-Visible spectrophotometer (Shimadzu). Quantity of eluted sensor molecules was estimated by normalizing absorbance readings with standard curves generated from known quantities of sensor molecules. Percentage values are obtained by normalizing released quantities with the original loaded quantity.
To evaluate sensor functionality following particle encapsulation and release, fluorescence signal from the collected supernatant was measured at the indicated time point (Genios FL plate reader (TECAN)), before and after the addition of the sensor molecule target. In particular, the different sensor molecules and their specific targets were incubated for 30 minutes before signal acquisition. The following are the sensor molecules and their targets: (1) CAM sensors with 1 U/ml esterases, (2) DAF-FM DA sensors with 62.5 μM SNAP, which spontaneously decomposes to generate NO (0.88 μM NO/minute), (3) β-actin MBs with 0.5 μM β-actin target sequence.
To evaluate sensor functionality following particle encapsulation and release, fluorescence signal from the collected supernatant was measured at the indicated time point (Genios FL plate reader (TECAN)), before and after the addition of the sensor molecule target. In particular, the different sensor molecules and their specific targets were incubated for 30 minutes before signal acquisition. The following are the sensor molecules and their targets: (1) CAM sensors with 1 U/ml esterases, (2) DAF-FM DA sensors with 62.5 μM SNAP, which spontaneously decomposes to generate NO (0.88 μM NO/minute), (3) β-actin MBs with 0.5 μM β-actin target sequence.
5
Spectrophotometric Analysis of Wine Color
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Wine chromatic parameters were measured with a Shimadzu UV-2450 UV–Visible spectrophotometer (Shimadzu Co., Kyoto, Japan) using 10 mm path length glass quartz cells and distilled water as a reference. The visible spectrum (400–700 nm) of wine was recorded at constant intervals (= 1 nm). Lightness (L*), red/green color coordinate (a*), yellow/blue color coordinate (b*), saturation (Cab*), and hue angle (Hab*) data were used to evaluate wine color using CIELab parameters (OIV, 2008 ). Each analysis was carried out three times.
6
Membrane-bound RH421 Absorbance Quantification
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UV-visible absorbance measurements were carried out with a UV-2450 UV-visible spectrophotometer (Shimadzu, Kyoto, Japan) using quartz semi-micro cuvettes. To reduce scattering contributions to the measured absorbance spectra of membrane-bound RH421, Na + ,K + -ATPase-containing membrane fragments, PLL or peptide fragments were added to both the reference and sample cuvettes. To further reduce any effects of scattering on the measured spectra, an ISR-2200 double-beam integrating sphere attachment (Shimadzu, Kyoto, Japan) was used. This reflects scattered light back into the photomultiplier and prevents it from being erroneously recorded as an absorbance. The bandwidth was 5 nm.
To quantify more precisely shifts in the absorbance spectrum of membrane-bound RH421 induced by interaction with PLL or peptide fragments, similar to the procedure outlined above for eosin, we utilised a ratiometric approach. In this case the absorbance ratio was determined by dividing the absorbance at 440 nm by the absorbance measured at 540 nm, i.e., R = A440/A540. This method eliminates any variation due to small differences in RH421 concentration.
To quantify more precisely shifts in the absorbance spectrum of membrane-bound RH421 induced by interaction with PLL or peptide fragments, similar to the procedure outlined above for eosin, we utilised a ratiometric approach. In this case the absorbance ratio was determined by dividing the absorbance at 440 nm by the absorbance measured at 540 nm, i.e., R = A440/A540. This method eliminates any variation due to small differences in RH421 concentration.
7
Enzymatic Uric Acid Conversion Assay
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Enzymatic activity measurements were carried out with a Shimadzu UV-2450 UV-visible spectrophotometer with a temperature-controlled cuvette-holder, at 30 °C. The activity was measured by following the decrease of absorbance of uric acid at 293 nm during the reaction. Uric acid was freshly prepared in 50 mM sodium borate buffer, pH 8.0. Kinetic constants were assayed by varying concentration of uric acid from 15 µM to 120 µM. The assay was performed in triplicate runs. Kinetic parameters were calculated by least-squares analysis from Lineweaver-Burk plots of substrate’s concentrations against reaction velocity. One unit of uricase activity is defined as the amount of enzyme required to convert 1 µmol of uric acid to allantoin per minute at 30 °C, pH 8.0.
8
Identification of Diosmin using UV and NMR
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Ultraviolet–visible (UV) spectroscopy and one-dimensional nuclear magnetic resonance (NMR) spectroscopy were used to identify diosmin. The UV spectrum of the purified compound was recorded in methanol using SHIMADZU UV-2450 UV-Visible Spectrophotometer in the range of 190–400 nm. Also, the 1H NMR and 13C NMR spectra of purified sample were recorded by a NMR spectrometer (Bruker Avance III 300 MHz, Germany) in hexadeuterodimethyl sulfoxide (DMSO-d6, 99.9 atom % D, ACROS Organics, USA) for determining the molecular structure. 1H NMR and 13C NMR spectra were recorded at 300 and 75 MHz, respectively [17 (link)].
9
Spectroscopic Characterization of Donor-Acceptor Dynamics
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The absorption measurements were performed with Shimadzu UV-2450 UV-Visible Spectrophotometer. We have used the buffer at various pH to create the baseline for absorbance measurement of the corresponding spectra. All the picosecond resolved fluorescence transients were measured by using commercially available time-correlated single-photon counting (TCSPC) setup with MCP-PMT from Edinburgh instrument, U.K. (instrument response function (IRF) of ∼80 ps) using a 409 nm excitation laser source. The details of the time-resolved fluorescence setup are identical to the previously reported article (23 (link)–28 (link)). A quartz cuvette of path length 1 cm was used for all the optical measurements. To estimate the Forster resonance energy transfer efficiency of the donor (EtBr) to the acceptor (TB) and hence to determine the donor-acceptor pairs we have followed the previously reported methodology. The donor-acceptor distance (r) can be calculated using the formula r6 = [R0 6 (1−E)/E], where R0 is the Forster’s distance and E is the efficiency of energy transfer. Here, the efficiency of energy transfer (E) is calculated from the lifetimes of the donor in the absence and presence of acceptors (τ D and τ DA) using the formula: E = 1-τ DA/τ D.
10
Sediment Properties Analysis Protocol
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A sediment properties analysis with ~10 g of fresh sediment was conducted at the Institute of Mountain Hazards and Environment, Chinese Academy of Sciences (Chengdu, Sichuan, China), according to Wang et al. (48 (link)). In brief, nitrate and ammonia were detected after 1 M HCl treatment followed by analysis with a colorimetric Auto-analyzer 3 (SEAL Analytical GmbH, Norderstedt, Germany). The concentrations of total carbon (TC) and total nitrogen (TN) were determined by overdrying the sediments at 105°C and then using a Vario Macro cube element analyzer (Elementar Analysensysteme GmbH, Langenselbold, Germany). Total phosphate (TP) was measured after digestion of the sediment with nitric-perchloric acid (49 (link)), using the molybdate colorimetric method with a Shimadzu UV-2450 UV-Visible spectrophotometer (Shimadzu Corp., Kyoto, Japan). Analysis of stable C and N isotopes was conducted in the Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences (Nanjing, Jiangsu, China), using the method described by Liu et al. (50 (link)).
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