Samples from the fibrillized α-syn aliquots or of the control α-syn monomers stored at 4°C were diluted to 0.1 mg/ml in 100 μl of phosphate-buffered saline (PBS) containing either 20 μM ThT, 20 μM X-34, 0.1% SYBR Green I (SG) commercial stock solution, or nothing else. The diluted samples were distributed either in 96-well plates (samples with probes) or in UVettes (Eppendorf) (samples without probe). After 20 min at room temperature protected from ambient light, the plates were read under orbital shaking in a BMG LABTECH FLUOstar OPTIMA fluorimeter. Excitation/emission wavelength pairs were 380 nm/520 nm for X-34, 450 nm/480 nm for ThT, and 485 nm/520 nm for SG. The UVettes were read at 280 nm using the 1-cm optical path with an Eppendorf biophotometer in absorbance mode to generate the A280 light attenuation readout.
Uvette
Manufactured by Eppendorf
Sourced in Germany, Austria, United Kingdom
The UVette is a cuvette designed for use in ultraviolet (UV) spectroscopy applications. It is made of UV-transparent plastic and is used to hold samples for measurement in a UV-Vis spectrophotometer.
Automatically generated - may contain errors
Lab products found in correlation
Dynapro nanostar, by Wyatt Technology (12 mentions)
Dynamics software, by Wyatt Technology (4 mentions)
Dynapro nanostar dynamic light scattering detector, by Wyatt Technology (3 mentions)
Dynamics 7, by Wyatt Technology (3 mentions)
Wyatt dynapro nanostar, by Wyatt Technology (2 mentions)
Prism 5, by GraphPad (2 mentions)
Gotaq dna polymerase, by Promega (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Qiaamp dna blood mini kit, by Qiagen (2 mentions)
Biophotometer, by Eppendorf (2 mentions)
F 4500 spectrofluorometer, by Hitachi (2 mentions)
Zero2, by Merck Group (2 mentions)
Temperature control unit, by Agilent Technologies (2 mentions)
Cary 3 uv vis spectrophotometer, by Agilent Technologies (2 mentions)
Uv 1800 spectrophotometer, by Eppendorf (2 mentions)
Zetasizer nano zs instrument, by Malvern Panalytical (1 mentions)
Enspire, by PerkinElmer (1 mentions)
Nano s, by Malvern Panalytical (1 mentions)
Homogentisic acid, by Merck Group (1 mentions)
Prism 6 for windows, by GraphPad (1 mentions)
48 protocols using uvette
1
Fluorometric Analysis of Fibrillized α-Synuclein
2
Characterization of Colloidal Nanoparticles
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Zeta potential and hydrodynamic diameter measurements were performed using a Zetasizer Nano ZS instrument (from Malvern Instruments Ltd., Worcestershire, UK), fitted with a He-Ne laser (λ = 633 nm) and a backscatter detector fixed at 173°. For zeta potential measurements, PBNP samples were diluted thirtyfold with a solution containing 0.9% NaCl in order to mimic physiological ion concentrations. The pH of the sample was adjusted by the addition of a 0.01 M NaOH solution. DLS measurement was performed in a W130i DLS instrument (Avid Nano Ltd., High Wycombe, UK) using low volume disposable cUVettes (UVette, Eppendorf Austria GmbH). The sample was diluted tenfold with ultrapure water and filtered through a 0.22 um membrane filter. Data were processed with iSize 2.0 software utilizing the CONTIN algorithm. DLS measurements were performed weekly for a period of 6 weeks to determine colloidal stability. Samples were stored at 4°C.
3
Antibiotic Susceptibility Profiling of Urine Samples
4
Determining Metal-Binding Affinities
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Our approach to determine
metal-binding affinities followed that described by Young and Xiao.59 (link) For each competition (eq 1 ), a master stock was prepared to contain
both competing ligands (L1 and L2) in Mops buffer (50 mM, pH 7.4).
Serial dilutions of the metal (M) were prepared separately in deionized
water. Exactly 135 μL of the master stock was dispensed into
an Eppendorf UVette and 15 μL of the appropriate metal stock
was added. Solution absorbances were recorded and used to calculate
concentrations of apo- and metalated forms of each
ligand. These concentrations were plotted against metal concentrations
and fitted in DynaFit86 (link) using binding models
as described in the text. The known association or dissociation constants
for all competitor ligands are listed inTable S1D
metal-binding affinities followed that described by Young and Xiao.59 (link) For each competition (
both competing ligands (L1 and L2) in Mops buffer (50 mM, pH 7.4).
Serial dilutions of the metal (M) were prepared separately in deionized
water. Exactly 135 μL of the master stock was dispensed into
an Eppendorf UVette and 15 μL of the appropriate metal stock
was added. Solution absorbances were recorded and used to calculate
concentrations of apo- and metalated forms of each
ligand. These concentrations were plotted against metal concentrations
and fitted in DynaFit86 (link) using binding models
as described in the text. The known association or dissociation constants
for all competitor ligands are listed in
5
Dynamic Light Scattering of VLP Formulations
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MS2-SA VLP was diluted in PBS to 100 μL such that there was 1 μg of SA in solution. VLP-S2 and VLP-S2mutS2’ were each diluted in PBS to 100 μL such that there was 5 μg of S2 in solution. Each 100 μL solution was then pipetted into a UVette (Eppendorf), which was inserted into a DynaPro NanoStar Dynamic Light Scattering detector (Wyatt Technology). Dynamics software (Wyatt Technology) brought the temperature of the measurement cell to 25 °C. The detector then proceeded with the measurement. Each measurement was the result of 10 acquisitions and was output as % Intensity, which could be converted to % Mass using the Isotropic Spheres model.
6
Dynamic Light Scattering and Absorbance Measurements
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Dynamic light scattering (DLS) measurements were performed on a Wyatt DynaPro NanoStar (Wyatt Technology, Santa Barbara, CA) instrument using a disposable cUVette (Eppendorf UVette, 220 to 1600 nm), and data were processed using Wyatt DYNAMICS V7 software. Each analysis involved an average of 10 measurements. The data were exported for final plotting using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA).
Absorbance measurements were taken on a PerkinElmer EnSpire multimode plate reader. Untreated Corning 96-well half area black flat bottom polystyrene microplates were used (ref. 3694).
Absorbance measurements were taken on a PerkinElmer EnSpire multimode plate reader. Untreated Corning 96-well half area black flat bottom polystyrene microplates were used (ref. 3694).
7
Genomic DNA and RNA Extraction
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Genomic DNA (gDNA) and RNA were extracted using, respectively, the QIAamp® DNA Blood Mini Kit (51104, Qiagen) and RNeasy Mini Kit (74104, Qiagen) following manufacturer’s instructions. All nucleic acids were quantified by the Eppendorf Biophotometer using Eppendorf single sealed cUVettes, UVette (952010051, Eppendorf UK Limited). gDNA was assessed to determine the genotypes of the different cell lines using the primers: 3LoxP U1 (5′-CATTGTGGTCCTTGGCACAG-3′), 3LoxP D1 (5′-AAGGGCGTTGTCTTCTCACC-3′) and Hyg U1 (5′-CTTGTATGGAGCAGCAGACG-3′). The PCR of gDNA was performed using GoTaq DNA polymerase (Promega) and 0.5 μM primers following the manufacturer’s indications. The PCR program was: 120 s at 95 °C followed by 33 cycles of 30 s at 95 °C, 60 s at 63 °C and 60 s at 72 °C.
8
Characterizing VLP-S Size by DLS
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VLP-S was diluted in PBS to a concentration of 0.05 μg/μL, added to a UVette (Eppendorf), and inserted into a DynaPro NanoStar Dynamic Light Scattering detector (Wyatt Technology). Ten acquisitions per sample were collected at 25 °C and displayed as % Mass with the Isotropic Spheres model.
9
Nanoparticle Size Determination by DLS
10
Fluorescent Analysis of Silk Fibroin
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Silk fibroin solutions (around 500 µL) were pipetted into a 10-mm-pathlength disposable cUVette (UVette, Eppendorf). Fluorescence spectra of the silk fibroin solution were obtained using an F4500 spectrofluorometer (Hitachi, Schaumburg, IL, USA). The scan rate was 60 nm/min. The excitation and emission slits were 5 and 10 nm, respectively. The voltage of the photomultiplier tube detector was 700 V. The excitation wavelength was 300 nm. The emission from 320 to 500 nm wavelength was collected.
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