FRET experiments were conducted following the protocol of Hessling et al.6 (link). Atto488 (donor) and Atto550 (acceptor) (ATTO-TEC GmbH) labelled yeast Hsp90-D61C (200 nM) was used. Measurements were performed in 40 mM HEPES (pH 7.5), 150 mM KCl, 5 mM MgCl2, 2 mM DTT in a Fluoromax 4 fluorescence spectrophotometer (Horiba Jobin Yvon) at 30 °C. Closing of Hsp90 was induced by addition of 2 mM AMP-PNP or 2 mM ATPγS. Chase experiments were performed with a tenfold excess (4 µM) of unlabelled WT yeast Hsp90 (Hsp82) to disrupt the FRET complex. Samples were incubated for 60 min at 30 °C prior to the addition of the unlabelled species. For determination of re-opening rates, closing was induced with 2 mM ATPγS. After the equilibrium was reached, a tenfold excess (20 mM) of ATP was added, to induce re-opening of the Hsp82 dimer. The data were analysed with Origin 8.0 and fitted using a mono-exponential equation:
Fluoromax 4 fluorescence spectrophotometer
Manufactured by Horiba
Sourced in Japan
The FluoroMax 4 is a fluorescence spectrophotometer designed for sensitive and accurate fluorescence measurements. It features a high-intensity xenon lamp, monochromators for excitation and emission wavelength selection, and a photomultiplier tube detector. The FluoroMax 4 is capable of performing qualitative and quantitative fluorescence analyses.
Automatically generated - may contain errors
Lab products found in correlation
Atto488, by ATTO-TEC (1 mentions)
Atto 550, by ATTO-TEC (1 mentions)
Uv 3600, by Shimadzu (1 mentions)
Jem 2100 transmission electron microscope, by JEOL (1 mentions)
Tcs sp5 confocal microscope, by Leica (1 mentions)
Tensor 27 spectrometer, by Bruker (1 mentions)
Axis ultra dld x ray photoelectron spectrometer, by Shimadzu (1 mentions)
Bca protein assay kit, by Takara Bio (1 mentions)
Octadecyl rhodamine b chloride r18, by Thermo Fisher Scientific (1 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated goat anti rabbit igg secondary antibody, by OriGene (1 mentions)
D max 2500, by Rigaku (1 mentions)
Jem 2010 transmission electron microscope, by JEOL (1 mentions)
U 3900 spectrophotometer, by Hitachi (1 mentions)
Fls980 spectrometer, by Edinburgh Instruments (1 mentions)
11 protocols using fluoromax 4 fluorescence spectrophotometer
1
FRET Analysis of Hsp90 Conformational Dynamics
2
Structural and Optical Characterization of Carbon Dots
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Transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) images were taken using a JEM-2100transmission electron microscope (Jeol, Japan). The normal TEM samples were prepared by dropping the aqueous solution of CDs onto copper grids and then dried under drying lamp. The statistical size distribution of CDs was obtained using the software of Nano Measurer 1.2 on the basis of TEM images (counting more than 200 dots for each sample in different images). Fourier transform infrared spectroscopy (FTIR) patterns were measured in range of 400–4000 cm−1 on a Nicolet 5700 FTIR spectrophotometer(Nicolet, USA). The X-ray photoelectron spectra (XPS) were recorded on a PHI 5000 Versa Probe electron energy spectrometer (UIVAC-PHI,Japan). Light absorption properties were obtained using ultraviolet–visible (UV–vis) spectrophotometer (UV-3600,Shimadzu, Japan). The measurement of PL was carried out on a Fluoromax-4 fluorescence spectrophotometer (Horiba, Japan). Fluorescence lifetime was measured with a time-resolved spectroscope FluoroLog 3-TCSPC (Horbia, Japan). The fluorescence microscope images were taken with a Leica TCS SP5 confocal microscope (Leica Microsystems, Germany).
3
Comprehensive Characterization of Carbon Quantum Dot Phosphors
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Transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM) images of CQDs/PC phosphor were obtained on JEOL JEM-2010. The powder sample was first dispersed in deionized water ultrasonically and then dripped onto ultra-thin carbon films. Fourier-transform infrared (FTIR) spectra were obtained on a Bruker Tensor 27 spectrometer with the sample in KBr disk. The X-ray diffraction (XRD) pattern of the CQDs/PC was measured by Rigaku-D/MAX 2500 diffractometer with Cu Kα (λ = 1.5406 Å) radiation at a scanning speed of 4°/min in the 2θ range from 10° to 80°. Photoluminescence (PL) spectra were recorded on Horiba Fluoromax-4 fluorescence spectrophotometer. The thermogravimetric (TG) curve was characterized by Setaram Labsys Evo TG analyzer under a high-purity argon atmosphere with a 10 K/min heating rate. The absolute QY of CQDs/PC phosphor was measured by an integrating sphere. X-ray photoelectron spectroscopy (XPS) measurement was conducted on Kratos AXIS ULTRA DLD X-ray photoelectron spectrometer with mono X-ray source Al Kα excitation. WLED devices were tested by the F-star photoelectric testing system.
4
Deubiquitination Activity Inhibition Assay
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Inhibition of the deubiquitination activities of the chUSP1FL control protein and mutant proteins was assessed using the irreversible DUB inhibitor ubiquitin vinyl sulfone (Ub-VS, Cat. No. U-212, Boston Biochem). 125 nM chUSP1FL protein or chUSP1FL/chUAF1 protein complex and 4 μM Ub-VS were preincubated in 1×reaction buffer for 1 hour at 37°C, before the deubiquitination reaction was initiated by the addition of Ub-AMC to a final concentration of 100 nM. Enzymatic activity was assessed by measuring the fluorescence intensity of the AMC released during the reaction using a FluoroMax 4 fluorescence spectrophotometer (HORIBA Scientific) with excitation and emission wavelengths of 380 nm and 460 nm, respectively. Kinetics were calculated as described below.
5
Deubiquitination Kinetics of chUSP1 Variants
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The kinetics of deubiquitination of chUSP1FL control and mutant variants of chUSP1 protein, alone or as part of the chUSP1/ chUAF1 complex, were assessed using Ub-AMC as a substrate. Deubiquitination reactions containing 0.1 to 1 μM Ub-AMC and proteins at the following concentrations were set up in 1× reaction buffer: 125 nM chUSP1FL, 20 nM chUSP1FL/chUAF1, 150 nM chUSP1H603A/chUAF1, 150 nM chUSP1D758A/chUAF1, 320 nM chUSP1C91S/chUAF1, 150 nM chUSP1C91A/chUAF1, 150 nM chUSP1H594A/chUAF1, or 150 nM chUSP1CH91,594SA/chUAF1. Preliminary experiments were performed to optimize the protein concentrations used in reactions and to ensure that their enzymatic activities were both comparable and within the detectable range. Enzymatic activity was assessed by measuring the fluorescence intensity of AMC released during the reaction using a FluoroMax 4 fluorescence spectrophotometer (HORIBA Scientific) with excitation and emission wavelengths of 380 nm and 460 nm, respectively, and enzyme kinetics were calculated as described below.
6
Viral Invasion of Midgut Epithelium
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To determine the role of BmSUH in viral invasion of midgut epithelial cells, fluorescence-dequenching assays were performed. To prepare ODVs, supernatants of OB lysates were purified by ultracentrifugation through 30%−60% (w/v) sucrose as previously described [60 (link)]. Subsequently, the purified ODV was labeled with the self-quenching fluorescent probe octadecyl rhodamine B chloride (R18; Invitrogen Life Technologies), as described previously [10 (link)]. Labeled ODVR was quantified using BCA protein Assay Kit (Takara) and stored in the dark at 4°C until use. Newly molted fourth-instar WT and ΔBmSUH larvae were inoculated orally with 3 μg labeled ODVR (n = 24 larvae) or PBS (n = 24 larvae). At 30-, 60-, and 90-min post-inoculation, after which the midgut of each larva was dissected and rinsed in 200 μL chilled separation buffer (100 mM NaCO3, 100 mM KCl, 100 mM EGTA, pH 9.5) and stored in the dark at −80°C until use. The amounts of ODV bound and fused were measured using FLUOROMAX-4 fluorescence spectrophotometer (Horiba Jobin Yvon) at excitation/emission wavelengths of 560nm/583nm, after which the relative fluorescence units per mg ODVR protein was calculated.
7
Characterization of Optical Properties
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Absorbance spectra were measured using a computer-controlled spectrophotometer (Thermo Nanodrop 2000c, USA). The fluorescence emission spectrum were obtained by FluoroMax-4 fluorescence spectrophotometer (HORIBA Jobin Yvon) using 600 nm as excitation wavelength, 620 nm to 750 nm as emission wavelength range and 5 nm as emission slit width.
8
Characterization of UL36(480) Hydrolysis Specificity
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SUMO1-GST, SUMO2-GST, SUMO3-GST [47 ,48 ], di-SUMO2(K11), or di-SUMO3(K11) and various rhodamine-conjugated ubiquitin-like proteins (Boston Biochem) were used as substrates for the characterization of hydrolysis specificity of 1 μM UL36(480). The reactions were performed as described above using SUMO substrate, and hydrolysis products were identified by Western blotting using primary antibody against SUMO1 (Cat#: ab5316) or SUMO2/3 (Cat#: ab3742) (dilution 1:2000, Abcam), and HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5000, OriGene) was used for ECL imaging. Hydrolysis specificity of UL36(480) on rhodamine-conjugated ubiquitin-like proteins was assessed in reactions containing 0.1μM of FAT10-Rhodamine, NEDD8-Rhodamine, UFM1-Rhodamine, or ISG15-Rhodamine substrates (Boston Biochem), respectively, and 1 μM UL36(480) in a final volume of 100 μL 1× reaction buffer at 37 °C. The fluorescence intensity of rhodamine group hydrolyzed from the reactions was monitored on a FluoroMax 4 fluorescence spectrophotometer (HORIBA Scientific, Edison, NJ, USA) with excitation and emission wavelengths of 570 and 590 nm, respectively.
9
Comprehensive Characterization of Materials
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TEM and HRTEM images were obtained with a JEOL JEM‐2010 transmission electron microscope. XRD patterns were carried out by X‐ray diffraction using Cu Kα radiation (Rigaku‐D/Max 2500). Raman spectra were recorded using a HORIB A Evolution Raman spectrometer. FTIR spectra were collected by a TENSOR2 spectrometer. XPS curves were obtained on an AXIS ULTRA DLD X‐ray diffractometer. UV–vis spectra were recorded using a HITACHI U‐3900 spectrophotometer. The PL spectra were collected on the HORIBA FluoroMax‐4 fluorescence spectrophotometer. The TRPL curves were measured on an EDINBURGH FLS980 spectrometer. The photoelectric properties of the LEDs were measured by Spectrascan PR655. The thermogravimetric curve was measured using a Setaram Labsys Evo thermogravimetric analyzer with a heating rate of 10 °C min−1 in a nitrogen atmosphere.
10
Fluorescence Spectroscopy of DNA Oligos
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Fluorescence spectra was performed with a HORIBA FluoroMax-4 fluorescence spectrophotometer (Japan) and a 1 cm path length quartz cuvette. The reaction samples containing 1 μM oligos were prepared in 10 mM LiCac buffer (pH 7.0) and 150 mM KCl/LiCl to form a total volume of 100 μl. The mixtures were vortexed and heated at 95°C for 5 min and allowed to cool for 15 min at room temperature for renaturation. ThT ligand (1 μM) was added to the mixture. The emission spectra were collected from 440 to 700 nm with an excitation wavelength of 425 nm. The entrance and exit slits were 5 and 2 nm respectively, with the data collected every 2 nm. All the data were analyzed with Microsoft Excel.
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