Circular dichroism (CD), low-temperature (77 K), and RT fluorescence were performed on thylakoid membrane fraction isolated as described in Garstka et al. (2007) (link) and diluted to Chl concentration of 10 μg mL−1. For CD measurements we followed the procedure described in Mazur et al. (2019) (link). The Chl emission spectra were recorded using modified Shimadzu RF-5301PC as described in Mazur et al. (2019) (link). The Chl fluorescence excitation spectra were recorded using FS5 Spectrofluorometer (Edinburgh Instruments) equipped with 77 K Optistat DN2 cryostat (Oxford Instruments). For membrane fluidity measurements, isolated thylakoids were diluted to Chl concentration of 2.5 μg mL−1 in 20-mM Hepes buffer (pH 7.5) containing 330-mM sorbitol. After 10 min at 25°C, sample was incubated with 1-µM laurdan for 30 min at 25°C. Steady-state fluorescence emission spectra were recorded (Shimadzu RF-5301PC) at 25°C in the range of 400–600 nm after excitation at 390 nm; excitation and emission slits were set to 10 and 5 nm, respectively. Generalized polarization values were calculated according to the formula presented in Figure 6G .
Rf 5301pc
Manufactured by Shimadzu
Sourced in Japan, United States, Italy
The RF-5301PC is a bench-top fluorescence spectrophotometer designed for general fluorescence measurements. It features a xenon lamp as the excitation light source and two monochromators to select the excitation and emission wavelengths. The instrument can measure fluorescence intensity, excitation and emission spectra.
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Lab products found in correlation
Uv 2450, by Shimadzu (12 mentions)
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Uv 1800, by Shimadzu (5 mentions)
Uv 2550, by Shimadzu (5 mentions)
Jem 2100, by JEOL (4 mentions)
Trichloroacetic acid, by Merck Group (4 mentions)
Nah2po4, by Merck Group (4 mentions)
F 4500, by Shimadzu (4 mentions)
D8 advance, by Bruker (3 mentions)
Jem 2100f, by JEOL (3 mentions)
Spectrofluorophotometer, by Shimadzu (3 mentions)
Carboxy h2dcfda, by Thermo Fisher Scientific (3 mentions)
Escalab 250, by Thermo Fisher Scientific (3 mentions)
Nicolet is50, by Thermo Fisher Scientific (3 mentions)
Reference 600 potentiostat galvanostat zra, by Gamry (2 mentions)
Uv 3600 plus, by Shimadzu (2 mentions)
S 4800, by Hitachi (2 mentions)
Tensor 27 system fourier transform infrared spectrometer, by Bruker (2 mentions)
Ht7700, by Hitachi (2 mentions)
Oleic acid, by Merck Group (2 mentions)
341 protocols using rf 5301pc
1
Characterization of Thylakoid Membranes
2
Cerebral Oxidative Stress Modulation
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Samples of the prefrontal cortex and hippocampus were collected to determine RS and TBARS levels. These measurements were performed to evaluate the effect of 4-PSQ on the modulation of cerebral oxidative stress.
RS levels were determined by a spectrofluorimetric method, using a 2′,7′ dichlorofluorescein diacetate (DCHF-DA) assay (Loetchutinat et al. 2005 (link)). Briefly, an aliquot of S1 was incubated with 1 mM DCHF-DA and 10 mM Tris–HCl pH 7.4. The oxidation of DCFH-DA to fluorescent dichlorofluorescein (DCF) is measured for the detection of intracellular RS. The DCF fluorescence intensity emission was recorded at 520 nm (with 480 nm excitation) (Shimadzu RF-5301 PC). RS levels were expressed as units of fluorescence.
TBARS content was used as a marker of lipid peroxidation (Ohkawa et al. 1979 (link)). An aliquot of S1 was added to the reaction mixture containing: thiobarbituric acid (0.8%), sodium dodecyl sulfate (8.1%), and acetic acid (pH 3.4) and incubated at 95 °C for 2 h. The absorbance was measured at 532 nm in a spectrophotometer (Shimadzu RF-5301 PC). Results were reported as nmol malondialdehyde (MDA)/mg protein.
RS levels were determined by a spectrofluorimetric method, using a 2′,7′ dichlorofluorescein diacetate (DCHF-DA) assay (Loetchutinat et al. 2005 (link)). Briefly, an aliquot of S1 was incubated with 1 mM DCHF-DA and 10 mM Tris–HCl pH 7.4. The oxidation of DCFH-DA to fluorescent dichlorofluorescein (DCF) is measured for the detection of intracellular RS. The DCF fluorescence intensity emission was recorded at 520 nm (with 480 nm excitation) (Shimadzu RF-5301 PC). RS levels were expressed as units of fluorescence.
TBARS content was used as a marker of lipid peroxidation (Ohkawa et al. 1979 (link)). An aliquot of S1 was added to the reaction mixture containing: thiobarbituric acid (0.8%), sodium dodecyl sulfate (8.1%), and acetic acid (pH 3.4) and incubated at 95 °C for 2 h. The absorbance was measured at 532 nm in a spectrophotometer (Shimadzu RF-5301 PC). Results were reported as nmol malondialdehyde (MDA)/mg protein.
3
Evaluating Ligand Binding Affinity to SaveOBP9
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A total of 30 ligands (Table 1 ) were tested in the fluorescence binding assay to assess the binding affinity of SaveOBP9 using a fluorescent probe, i.e., N-phenyl-1-naphthylamine (1-NPN). Methanol (spectrophotometric-grade) was used to prepare the standard solution of each tested ligand (Sigma Aldrich, St. Louis, MO, USA). The binding ability of 1-NPN was measured by adding 1-NPN (1 mM) into protein solution (2 μM L−1) diluted with Tris-HCl (30 mM), and the final concentration was made to 0 to 20 μM L−1 at room temperature. The blend of SaveOBP9/1-NPN was excited at a precise wavelength (337 nm), and discharge spectra ranging from 360 to 600 nm were recorded with a scanning speed of 300 nm min−1 using a RF-5301PC fluorescence spectrophotometer (RF-5301PC, Shi-madzu, Kyoto, Japan) with a slit width of 10 nm and a light path quartz cuvette (1 cm) at a temperature of 25 °C.
The ligand’s binding with SaveOBP9 was conducted with three independent replicates. The ligand’s binding affinity (Ki) of SaveOBP9 was calculated as follows:
where IC50 is the ligand’s concentration, [1-NPN] is the free concentration of 1-NPN, and K1-NPN is the dissociation constant (Kd) of the complex, SaveOBP9/1-NPN.
The ligand’s binding with SaveOBP9 was conducted with three independent replicates. The ligand’s binding affinity (Ki) of SaveOBP9 was calculated as follows:
where IC50 is the ligand’s concentration, [1-NPN] is the free concentration of 1-NPN, and K1-NPN is the dissociation constant (Kd) of the complex, SaveOBP9/1-NPN.
4
Fluorescent Graphene Oxide Preparation
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Fluorescein isothiocyanate-coated graphene oxide (FTIC-GO) was prepared and used to evaluate GO penetration into the embryo as described by Cheng et al. (2015) , and preliminary tests were carried out to establish the best preparation conditions. The final sample, for use in the bioassay, was prepared by mixing 5 mL of the solution OG 1 g.L -1 , 50 μL of the solution FITC 1 g.L -1 , e 5 mL of ultrapure water. The mixture was incubated for 20 min in the dark, sonicated for 4 min, and then dialyzed to remove the free FTIC (Inlab 133 membrane, 24-porosity). Dialysis was carried out for approximately 4 h, with periodic changes of the dialysis water and monitoring of the fluorescence emission (Shimadzu RF-5301 PC, λ excitation = 498 nm, λ emission = 516 nm). The fluorescence of the dialysis water fell from 5.3 to 0.09 at the end of the period. The labeling of the GO-FTIC sample was confirmed by spectrofluorimetry (Shimadzu RF-5301 PC, λ excitation = 498 nm, λ emission = 516 nm) and fluorescence microscopy. The fluorescence of the GO-FTIC samples was monitored and shown to be stable for up to 4 days after preparation.
5
Mitochondrial Calcium Retention Capacity
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The mitochondrial CRC was determined with fluorescent Ca 2+ -sensitive indicator calcium green-5N (Molecular Probes, Invitrogen, Eugene, OR USA) using a spectrofluorimeter Shimadzu RF-5301-PC (Shimadzu Corporation, Kyoto, Japan) at excitation/emission wavelengths of 506/535 nm (Gomez et al. 2008 , Singh et al. 2006) . The isolated mitochondria (1 mg protein/ml) were incubated at 25 °C in the buffer used for the measurement of respiration, the concentration of calcium green was 100 nmol/l. After 2-min incubation, 100 nmol CaCl 2 was added every 3 min, causing fluorescence flashes that decreased slowly as a result of mitochondrial Ca 2+ uptake. The CaCl 2 solution was added until a massive increase in fluorescence indicated a cessation of Ca 2+ uptake and MPTP opening. The mitochondrial CRC was calculated as a maximum amount of Ca 2+ accumulated in mitochondria and expressed as nmol/mg protein (Gomez et al. 2008) .
6
Mitochondrial Membrane Potential Measurement
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ΔΨ was measured by a spectrofluorimeter Shimadzu RF-5301-PC (Shimadzu Corporation, Kyoto, Japan) by transmembrane distribution of the fluorescence indicator tetramethylrhodamineethyl ester (TMRE, Molecular Probes, Invitrogen, Eugene, USA) (Paillard et al. 2009 , Scaduto and Grotyohann 1999 , Singh et al. 2006) . The excitation wavelength λEx was 550 nm, the emission wavelength λEm was 575 nm. The reaction was initiated by the addition of a mitochondrial suspension (2 mg protein) to a cuvette of the spectrofluorimeter to a 3 ml buffer containing 200 mM sucrose, 10 mM Tris-HCl, 5 mM KH 2 PO 4 , 0.01 mM EGTA, 2.5 mg/ml BSA fatty acid free, 3 mmol/l pyruvate and 3 mmol/l malate (pH 7.37, 25 °C), 40 nmol/l TMRE. The value of ΔΨ was estimated from the fall in fluorescence intensity after addition of 100 nmol/l FCCP to the incubation medium. The magnitude of the mitochondrial transmembrane potential was presented as the fluorescence intensity Vol. 68 difference before and after addition of FCCP and expressed as a percentage of the mitochondrial fluorescence of the intact (not subjected to I/R) heart in terms of mg protein of the mitochondria suspension.
7
Characterization of Gold Nanoclusters on Mesoporous Silica Nanoparticles
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The particle sizes and zeta potential were measured using a nanoparticle analyser SZ‐100 (Horiba Scientific, Kyoto, Japan). Inductively coupled plasma atomic emission spectroscopy (ICP‐OES) was used to analyse the quantities of AuNCs on the MSN@AuNCs and to determine the concentrations of the two nanoparticles. Ultraviolet‐visible (UV‐vis) absorption spectra were assessed on a Mettler Toledo UV5Nano (Mettler‐Toledo Ltd., Leicester, UK). The morphology and compositional distribution of AuNCs, MSNs and MSN@AuNCs were carried out using a JEM‐2100PLUS Transmission Electron Microscope (TEM). The fluorescence spectrophotometer (Shimadzu RF‐5301PC, Tokyo, Japan) was used to record the fluorescence spectra and the fluorescence decay behaviour of AuNCs and MSN@AuNCs. The fluorescence stability of AuNCs, MSN@AuNCs and Rhodamine were also assessed using the fluorescence spectrophotometer.
8
Quantifying Encapsulation Efficiency of NLS-Dox and anti-miR21 in HMNs
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To determine the efficiency of NLS-Dox and anti-miR21 encapsulation in the HMNs, 10 μL of 20% Triton X-100 (v/v) and 20% heparin (w/v) were added to 100 μL of fractionated samples and incubated for 30 minutes at ambient temperature. The incubation completely released the NLS-Dox and FAM from the lipid-coated nanoparticles or HMNs. Then, the solution was diluted with complexation buffer, producing appropriate dilutions for fluorescence determination. The fluorescence intensities of NLS-Dox and FAM were measured using spectrofluorometry (RF-5301PC; Shimadzu, Kyoto, Japan) at
of 470/590 nm and 490/518 nm, respectively. Concentration was calculated by interpolation using a standard curve. Encapsulation efficiency was calculated as the percentage of the NLS-Dox incorporated into the nanoparticles or HMNs relative to the initial total amount of the NLS-Dox, as follows:
and anti-miR21 encapsulation efficiency was also determined by this equation.
of 470/590 nm and 490/518 nm, respectively. Concentration was calculated by interpolation using a standard curve. Encapsulation efficiency was calculated as the percentage of the NLS-Dox incorporated into the nanoparticles or HMNs relative to the initial total amount of the NLS-Dox, as follows:
and anti-miR21 encapsulation efficiency was also determined by this equation.
9
Quantitative GUS Activity Assay
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Quantitative GUS activity was measured according to the method described by Jefferson et al. [17 (link)] with some modifications. Briefly, the shoots or roots of 14-days seedlings that carried different fragments of OP1 were homogenized in GUS extraction buffer (50 mM PBS, pH 7.0, 10 mM EDTA, pH 8.0, 20% methanol, 0.1% Triton X-100, 0.1% sodium lauryl sarcosine, and 10 mM β-mercaptethanol). Crude protein extract (50 μl) was added to 450 μl of extraction buffer containing 2 mM 4-methylumbelliferyl-β-D-glucuronide (MUG) at 37 °C for 30 min or 60 min, and thereafter 200 μl of the reaction mixture was added to 800 μl of stop buffer (0.2 M Na2CO3). The 4-methylumbelliferone fluorescence was measured using a spectrofluorophotometer (RF-5301PC, Shimadzu) at 460 nm with excitation at 355 nm. Protein concentration was quantified by methods described by Bradford [18 (link)]. GUS activity was calculated as pmol of 4-methylumbelliferon (4-MU) min per minute and per milligram of total soluble proteins and presented as GUS activity relative to the VC.
10
Characterization of Dye-Sensitized Solar Cells
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Absorption spectroscopy was carried out with UV-3600 Plus from Shimadzu, MD, USA. Emission spectroscopy was measured with RF-5301PC from Shimadzu, MD, USA. TiO2 paste was printed on FTO glass using WS-650 Series Spin Processor from Laurell Technologies Corporation, PA, USA. Carbon paint used in making cathode slides was purchased from Ted Pella, Inc., USA. The cell performance was measured using a 150 W fully reflective solar simulator with a standard illumination of air-mass 1.5 global (AM 1.5 G) having an irradiance of 100mW/cm2 (Sciencetech, Inc.) from London, Ontario, Canada. Reference 600 Potentiostat/Galvanostat/ZRA was purchased from GAMRY Instruments (Warminster, PA).
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