Human amidated NPY was labeled with Alexa Fluor 488 according to the manufacturer's protocol (Invitrogen, Carlsbad, CA, USA). The labeled product was isolated from free dye by gel filtration chromatography on a Bio‐gel P2 column and quantitatively determined by absorption spectrophotometry at wavelengths 280 and 494 nm, the latter of which is the maximum wavelength of free dye. The degree of labeling was calculated from the absorbance of the conjugate solution at 280 and 494 nm, as described by the manufacturer. The analysis of the labeling peptide was performed by Tricine SDS/PAGE. In brief, an aliquot of the labeled peptide was treated in a buffer containing 1% SDS and 1% β‐mercaptoethanol, without heating, prior to analysis by Tricine SDS/PAGE (16.5% T, 6% C resolving gel; 4% T, 3% C stacking gel). The fluorescence of the labeled peptide was observed under UV light and peptide on the gel was detected by staining with Coomassie Brilliant Blue R‐250. The fluorescence spectrum of the labeled amidated NPY was determined using an RF‐1501 spectrofluorophotometer (Shimadzu, Kyoto, Japan) and then compared to the labeled peptide after the cleavage by TTR.
Rf 1501 spectrofluorophotometer
Manufactured by Shimadzu
Sourced in Japan, United Kingdom, United States
The RF-1501 spectrofluorophotometer is a laboratory instrument manufactured by Shimadzu. It is designed to measure the fluorescence emission of samples across a range of wavelengths.
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Lab products found in correlation
Bovine serum albumin (bsa), by Bio-Rad (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Bradford method, by Bio-Rad (1 mentions)
400 mhz nmr spectrometer, by Agilent Technologies (1 mentions)
Spectrum two ft ir spectrophotometer, by PerkinElmer (1 mentions)
1601 spectrophotometer, by Schimadzu (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Drx 300 avance spectrometer, by Bruker (1 mentions)
Cary 50 spectrophotometer, by Agilent Technologies (1 mentions)
11 protocols using rf 1501 spectrofluorophotometer
1
Fluorescent Labeling and Analysis of Amidated NPY
2
Fluorometric Assay of NAGLU Activity
3
Fluorometric Rb Analysis in Milk
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The Rb concentration in milk was analyzed using the fluorometric method (AOAC method 970.65; Bradley, 2000; Webster et al., 2009) and was measured on a Shimadzu RF-1501 spectrofluorophotometer (Shimadzu Scientific Instrument Inc., Columbia, MD). Samples were prepared by adding 0.01 N hydrochloric acid to milk (10 mL) to pH between 5.0 and 6.0. Hydrochloric acid (10 N, 0.1 mL) was added and the sample was autoclaved under pressure (117 kPa) for 30 min at 121 to 123°C. Samples were cooled and pH adjusted between 6.0 and 6.5 using 0.025 N sodium hydroxide. Hydrochloric acid (0.1 N) was immediately added to stop further precipitation around pH of 4.5. Each sample was then filtered using 25 mm syringe with a 0.2-μm filter (Grace, Deerfield, IL) and 10-mL Norm-Ject syringe (Henke Sass Wolf Inc., Tuttlingen, Germany) before being measured using the spectrofluorophotometer.
4
Determining Critical Micelle Concentration using Pyrene Fluorescence
5
Determination of Critical Micelle Concentration
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Critical micelle concentration (CMC) was determined using pyrene as a fluorescence probe for the environment. 12 (link) Briefly, serial dilutions of different block copolymer micelle solutions were prepared in water (0.006 -50 μM) in amber vials. Pyrene in acetone was added to each polymer solution to achieve a final concentration of 2 μM. Micelles were agitated overnight, protected from light to allow for partitioning of pyrene into micelles and evaporation of acetone. Fluorescence was measured on a Shimadzu RF 1501 spectrofluorophotometer using an excitation wavelength of 333 nm and scanning emission wavelengths of 350 nm to 400 nm. The ratio of the fluorescent intensities at the 373 and 388 nm wavelengths (I 388 /I 373 ) was determined for each concentration of polymer. The CMC value was determined from the value of the inflection point of the curve. 13 (link)
6
Iduronidase Activity and GAG Quantification
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For biochemical assays, organs were weighed (wet weight) and homogenized in 3 volumes of PAD buffer (10 mM sodium phosphate, pH 5.8, 0.02% sodium azide, 0.1 mM dithiothreitol, and 0.1% Triton X-100). Iduronidase activity was assessed using 250 μmol/L 4-methylumbelliferyl α-l -iduronide substrate (4-MUI) (Glycosynth, Warrington, Cheshire, UK), as previously described, but with a modified incubation temperature (37°C) and incubation time (30 min).23 (link) Net fluorescence was determined by RF-1501 spectrofluorophotometer (Shimadzu, Columbia, MD) at the wavelength of 365 nm and 440 nm for excitation and emission, respectively. One activity unit is defined as the activity catalyzing the hydrolysis of 1 nmol substrate in 1 hr at 37°C. Protein concentrations in the extracts were determined by the Bradford method (Bio-Rad, Irvine, CA). Results were expressed as U/mg of protein. GAG amount in tissue was assessed using the carbazole reaction with glucuronic acid as the standard.24 Tissue results were expressed as nmol GAG (glucuronic acid) per mg protein.
7
Characterization of Functionalized Polycaprolactone
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1H NMR spectra were recorded on an Agilent 400 MHz NMR spectrometer at room temperature. Fourier transform infrared (FT-IR) spectra were measured with a Perkin-Elmer Spectrum Two FT-IR spectrophotometer. Number and weight average molecular weights (Mn and Mw) and molecular weight distributions (Mw/Mn) were determined by gel permeation chromatography (GPC) using an Agilent GPC Instrument (Model 1100) consisting of a pump, a refractive index detector and two Waters Styragel columns (HR 5E, HR 4E), using THF as the eluent at a flow rate of 0.5 mL/min at 23 °C and toluene as an internal standard. Molecular weights were calculated by using monodisperse polystyrene standards. UV–vis spectra were registered on a Schimadzu 1601 spectrophotometer. Fluorescence spectra were recorded on a Shimadzu RF-1501 spectrofluorophotometer. Thermal stabilities and the glass transition temperatures of the polymers were investigated on a Perkin-Elmer TGA/DTA 7300 thermal analysis systems, under N2 flow with a heating rate of 20 °C/min. Molecular weights of PCL-CH, PCL-(Br)2, PCL-(N3)2 and PCL-(PI)2 were calculated with the aid of polystyrene standards by using the following conversion formula [35 (link)]: MPCL = 0.259 MPSt1.073.
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1H NMR spectra were recorded on an Agilent 400 MHz NMR spectrometer at room temperature. Fourier transform infrared (FT-IR) spectra were measured with a Perkin-Elmer Spectrum Two FT-IR spectrophotometer. Number and weight average molecular weights (Mn and Mw) and molecular weight distributions (Mw/Mn) were determined by gel permeation chromatography (GPC) using an Agilent GPC Instrument (Model 1100) consisting of a pump, a refractive index detector and two Waters Styragel columns (HR 5E, HR 4E), using THF as the eluent at a flow rate of 0.5 mL/min at 23 °C and toluene as an internal standard. Molecular weights were calculated by using monodisperse polystyrene standards. UV–vis spectra were registered on a Schimadzu 1601 spectrophotometer. Fluorescence spectra were recorded on a Shimadzu RF-1501 spectrofluorophotometer. Thermal stabilities and the glass transition temperatures of the polymers were investigated on a Perkin-Elmer TGA/DTA 7300 thermal analysis systems, under N2 flow with a heating rate of 20 °C/min. Molecular weights of PCL-CH, PCL-(Br)2, PCL-(N3)2 and PCL-(PI)2 were calculated with the aid of polystyrene standards by using the following conversion formula [35 (link)]: MPCL = 0.259 MPSt1.073.
8
Fluorometric Assay for β-Galactosidase Activity
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Endothelial cells or plasma β-Galactosidase activity was measured performing a fluorometric assay using 1mM 4-Methyl-umbelliferyl-β-D-galactopyranoside in citrate-phosphate buffer, pH 4.0 as substrate.
Endothelial cells pellet was re-suspended in a suitable amount of water and sonicated 3 times for 15” in ice-bath to obtain homogenous cells suspension. A quantitative estimation of total protein concentration was made using a colorimetric assay (Bio-Rad Protein Assay,) reading the optical density (OD) at 595 nm [48 (link)].
50 μL of plasma samples and a variable volume of cells suspension (μL) containing 20-30 μg of proteins, were incubated with 200 μL and 300 μL of the substrate solution, respectively. After 1 hour at 37°C, the reaction was stopped with 2.5 mL of glycine-carbonate buffer. The fluorescence of the liberated 4-Methylumbelliferone was measured on RF-1501 spectrofluorophotometer (Shimadzu) at excitation wavelength=360 nm and at emission wavelength= 446 nm.
Enzymatic activity was expressed in nM/mg/hr for endothelial cells and nM/ml/hr for plasma samples respectively [49 (link)].
Endothelial cells pellet was re-suspended in a suitable amount of water and sonicated 3 times for 15” in ice-bath to obtain homogenous cells suspension. A quantitative estimation of total protein concentration was made using a colorimetric assay (Bio-Rad Protein Assay,) reading the optical density (OD) at 595 nm [48 (link)].
50 μL of plasma samples and a variable volume of cells suspension (μL) containing 20-30 μg of proteins, were incubated with 200 μL and 300 μL of the substrate solution, respectively. After 1 hour at 37°C, the reaction was stopped with 2.5 mL of glycine-carbonate buffer. The fluorescence of the liberated 4-Methylumbelliferone was measured on RF-1501 spectrofluorophotometer (Shimadzu) at excitation wavelength=360 nm and at emission wavelength= 446 nm.
Enzymatic activity was expressed in nM/mg/hr for endothelial cells and nM/ml/hr for plasma samples respectively [49 (link)].
9
Characterization of Synthesized Polymers
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To study the functional groups of the compounds, the Fourier transform infrared (FT-IR) spectra were measured by means of a Perkin Elmer FT spectrum RX1 (USA). To investigate the inherent viscosities (ηinh) of synthesized polymers, a solution of 0.5 g dL−1 of HPs in DMAc was prepared, and then an Ubbelohde suspended-level viscometer was used to measure their viscosities at 30°C.
The monomer and polymer structures were studied by recording their solution state 1 H NMR (300 MHz) and 13 C NMR (75 MHz) spectra on a Bruker DRX 300 AVANCE spectrometer (Germany) using deuterated dimethyl sulfoxide and trimethylsilane (TMS) as solvent and reference, respectively.
A Du Pont 2000 thermal analysis system (Mettler Toledo-Switzerland) was used for thermo-gravimetric analysis (TGA) under N2 atmospheres at a heating rate of 10°C min−1. Glass transition temperatures (Tg) were investigated by a differential scanning calorimetry (DSC-2010 model) thermal analysis (Mettler Toledo-Switzerland) apparatus at a heating rate of 10°C min–1. A Shimadzu RF-1501 spectrofluorophotometer was used to obtain the fluorescence spectra.
The monomer and polymer structures were studied by recording their solution state 1 H NMR (300 MHz) and 13 C NMR (75 MHz) spectra on a Bruker DRX 300 AVANCE spectrometer (Germany) using deuterated dimethyl sulfoxide and trimethylsilane (TMS) as solvent and reference, respectively.
A Du Pont 2000 thermal analysis system (Mettler Toledo-Switzerland) was used for thermo-gravimetric analysis (TGA) under N2 atmospheres at a heating rate of 10°C min−1. Glass transition temperatures (Tg) were investigated by a differential scanning calorimetry (DSC-2010 model) thermal analysis (Mettler Toledo-Switzerland) apparatus at a heating rate of 10°C min–1. A Shimadzu RF-1501 spectrofluorophotometer was used to obtain the fluorescence spectra.
10
Spectroscopic analysis of cyanine dyes
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Absorbance spectra were measured using a Cary 50 spectrophotometer (Varian, Palo Alto, CA, USA) interfaced to a PC, with a spectral bandwidth of 2 nm. Fluorescence spectra for the pentamethine cyanine dyes were obtained using a Shimadzu RF-1501 Spectrofluorophotometer (Shimadzu Scientific Instruments, Columbia, MD, USA) interfaced to a PC, with the spectral bandwidths for both excitation and emission set to 10 nm and the sensitivity set to high.
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