EPR measurements of •O2− generation within the SMP preparation were carried out on a Bruker EMX Micro spectrometer operating at 9.43 GHz with 100 kHz modulation frequency at room temperature [13 ]. The reaction mixture containing the NADH-linked respiration buffer supplemented with DTPA (1 mM), NADH (0.5 mM), and DMPO (90 mM) was mixed with the SMP preparation (to a final 1.0 mg protein/mL) at 30 °C for 4 min. The reaction mixture was then transferred into a 50-μL capillary (Drummond Wiretrol, Broomall, PA), sealed, loaded into the EPR resonator (HS cavity, Bruker Instrument, Billerica, MA), equilibrated to 298 K, and tuned within 2 min. The scan of the EPR spectrum was started at exactly 6 min after the initial reaction. Parameters: center field 3360 G, sweep width 100 G, power 20 mW, receiver gain 1 × 105, modulation amplitude 1 G, conversion time 40.96 ms, time constant 163.84 ms, number of scans: 5. The spectral simulations were performed using the WinSim program developed at NIEHS by Duling [14 (link)].
Emxmicro spectrometer
Manufactured by Bruker
Sourced in Germany
The EMXmicro spectrometer is a compact and versatile electron paramagnetic resonance (EPR) spectrometer designed for a wide range of applications. It provides high-sensitivity detection and a user-friendly interface for researchers and scientists who require reliable EPR analysis capabilities.
Automatically generated - may contain errors
Lab products found in correlation
Hs cavity, by Bruker (2 mentions)
Er4119hs resonator, by Bruker (2 mentions)
Annexin 5 magnetic microbeads kit, by Miltenyi Biotec (1 mentions)
Elexsys e580 spectrometer, by Bruker (1 mentions)
Xepr software package, by Bruker (1 mentions)
2600 uv vis recording spectrophotometer, by Shimadzu (1 mentions)
Esr 900 flow cryostat, by Oxford Instruments (1 mentions)
Emxnano spectrometer, by Bruker (1 mentions)
Er 4122 shq resonator, by Bruker (1 mentions)
Itc 502 temperature controller, by Oxford Instruments (1 mentions)
Er 4119hs, by Bruker (1 mentions)
E580 spectrometer, by Bruker (1 mentions)
Xsophe, by Bruker (1 mentions)
Neo 400 mhz spectrometer, by Bruker (1 mentions)
Ammonium metavanadate nh4vo3, by Merck Group (1 mentions)
D mannitol c6h14o6, by Merck Group (1 mentions)
Vertex 70 spectrometer, by Bruker (1 mentions)
Esr900 cryostat, by Oxford Instruments (1 mentions)
Itc503s temperature controller, by Oxford Instruments (1 mentions)
26 protocols using emxmicro spectrometer
1
EPR Spectroscopy of Superoxide Formation
2
Quantifying Apoptosis in Infarcted Mouse Heart
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To quantify total apoptosis in the infarcted tissue of mouse heart, we modified an assay method originally developed by Fabisiak et al. [49 (link)], using annexin-V magnetic microbeads kit from Miltenyi Biotec GmbH, Germany (Cat. No. 130-090-201). After sham or IR surgery, the heart was quickly isolated from mice, cannulated via aortic arch and perfused with ice cold saline followed by 1x annexin-V binding buffer supplied by the manufacturer. Following complete removal of circulating blood, the heart was perfused with 250 μL of annexin-V microbead suspension and incubated at 2-4°C for 20 minutes. At the end of the incubation period, the heart was perfused with ice cold 1x annexin-V binding buffer and the entire infarcted tissue (area lower to the occlusion site in the LAD) was dissected out. Total annexin-V bound to infarcted tissue was quantified by measuring conjugated iron spins using Bruker EMX Micro spectrometer at room temperature. EPR spectra were acquired under following scan conditions: microwave frequency, 9.83 GHz; power, 30 mW; attenuation 8 dB; modulation frequency, 100 kHz; modulation amplitude, 4.00 G; sweep time, 60 s; time constant, 20.48 s; receiver gain, 20 dB; magnetic field, 2110-4110 G. Absolute spin counts from spectra were calculated using Quantitative EPR module of Bruker Xenon Micro 1.3 software.
3
EPR Spectroscopy of Photocatalytic Reactions
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Continuous-wave EPR spectroscopy was carried out at X-band (9.85 GHz) using an EMX micro-spectrometer (Bruker). EPR data were collected at room temperature with a modulation amplitude of 10 G, with a microwave frequency of ~9.85 GHz and power of ~6.325 mW. Strong pitch (g = 2.0028) was used as a reference. Theoretical modelling of EPR spectra was performed using the EasySpin toolbox (Version 5.2.24) in Matlab58 (link).
All reagents were deoxygenated under Ar. In a typical experiment, DMPO (176.7 mmol/L) was dissolved in CH3CN and used as a spin trap. Acetophenone (0.050 mmol) and Na2SO3 (0.25 M) were dissolved in a mixed solution of CH3CN/H2O (1.5 mL/1.5 mL) and MFM-300(Cr) (0.005 mmol) was added. 0.2 mL of the resultant mixture was injected into an Ar deoxygenated vial, followed by 0.1 mL of the DMPO solution. 0.1 mL of the resultant DMPO mixed solution was then transferred into a capillary for freeze pumping to further degas the solution in order to remove all the dissolved gases (<0.01 mbar). The capillary was then directly used for EPR measurements. EPR spectra of the system were collected before (dark) and after (light) radiation (350–780 nm) by a Xe lamp for 10 min.
All reagents were deoxygenated under Ar. In a typical experiment, DMPO (176.7 mmol/L) was dissolved in CH3CN and used as a spin trap. Acetophenone (0.050 mmol) and Na2SO3 (0.25 M) were dissolved in a mixed solution of CH3CN/H2O (1.5 mL/1.5 mL) and MFM-300(Cr) (0.005 mmol) was added. 0.2 mL of the resultant mixture was injected into an Ar deoxygenated vial, followed by 0.1 mL of the DMPO solution. 0.1 mL of the resultant DMPO mixed solution was then transferred into a capillary for freeze pumping to further degas the solution in order to remove all the dissolved gases (<0.01 mbar). The capillary was then directly used for EPR measurements. EPR spectra of the system were collected before (dark) and after (light) radiation (350–780 nm) by a Xe lamp for 10 min.
4
EPR Spectroscopy of Radical Species
5
EPR Analysis of RNA Folding
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For the EPR sample preparation, the RNA was resuspended into folding buffer (10 mM HEPES, 10 mM KCl, 10 mM MgCl2; pH 7.5) in the absence or presence of 40 mM Gdm+. The samples were incubated at 95°C for 5 min and subsequently cooled on ice for 10 min. In the following, the Gdm+ concentrations are abbreviated as ?–Gdm+ (no Gdm+), +Gdm+ (0.4 mM Gdm+) and ++Gdm+ (40 mM Gdm+). cw X-Band EPR spectra were recorded on an EMXmicro spectrometer from Bruker BioSpin (Rheinstetten, Germany). The spectra were measured with a microwave power of 2 mW, a modulation frequency of 100 kHz, a modulation amplitude of 1 G, a microwave frequency of 9.6 GHz, and 1300 points in the field interval of 337 - 350 mT (Supplementary Figure S3 and Supplementary Table S5 ).
6
Extracellular ROS Trapping and EPR Analysis
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For trapping
extracellular ROS, we used the spin trap [5-(diisopropoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide;
2-diisopropylphosphono- 2-methyl-3, 4-dihydro-2H-pyrrole-1-oxide]
(DIPPMPO) (Enzo Life Science). Cells were harvested to give a final
concentration of 2 × 106 cells per assay. Stock solutions
of DIPPMPO were prepared in PBS at concentrations of 1 M. These solutions
were degassed with oxygen free nitrogen to remove oxygen from the
solution. RAW 264.7 cells were harvested to give an assay of 2 ×
106 cells which were incubated with the DIPPMPO spin trap
(final concentration of 100 mM) for 15 min prior to running EPR. For
LPS studies, a final concentration of 1 μg/mL was added and
cells were appraised by EPR. EPR spectra were run on a Bruker EMX-Micro
spectrometer (Bruker, UK), operating at 9 GHz utilizing gas-permeable
tubing for the cell samples and run at 37 °C. Spectra were analyzed
with WinEPR software to obtain identification of the radical adducts
trapped.
extracellular ROS, we used the spin trap [5-(diisopropoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide;
2-diisopropylphosphono- 2-methyl-3, 4-dihydro-2H-pyrrole-1-oxide]
(DIPPMPO) (Enzo Life Science). Cells were harvested to give a final
concentration of 2 × 106 cells per assay. Stock solutions
of DIPPMPO were prepared in PBS at concentrations of 1 M. These solutions
were degassed with oxygen free nitrogen to remove oxygen from the
solution. RAW 264.7 cells were harvested to give an assay of 2 ×
106 cells which were incubated with the DIPPMPO spin trap
(final concentration of 100 mM) for 15 min prior to running EPR. For
LPS studies, a final concentration of 1 μg/mL was added and
cells were appraised by EPR. EPR spectra were run on a Bruker EMX-Micro
spectrometer (Bruker, UK), operating at 9 GHz utilizing gas-permeable
tubing for the cell samples and run at 37 °C. Spectra were analyzed
with WinEPR software to obtain identification of the radical adducts
trapped.
7
Electron Spin Resonance Spectroscopy Methods
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Pulsed and continuous-wave (CW) ESR experiments were performed on a Bruker ElexSys E580 spectrometer coupled to a dielectric resonator (MD5), and additional CW-ESR experiments were performed on a Bruker EMXmicro spectrometer coupled to a Super High-Q cavity. Both spectrometers operate at X-band frequency (νmw = 9.8 GHz). CW-ESR spectra were recorded with magnetic field modulation amplitude and frequency of 0.1 mT and 100 kHz, respectively. The W-band CW-ESR spectra were recorded on a home-built spectrometer based on a Krymov bridge and probe43 (link), operating at a frequency νmw = 94.90 GHz, with a modulation amplitude of 0.1 mT and modulation frequency of 10 kHz.
8
Electron Paramagnetic Resonance of Photoactive Complex
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All electron paramagnetic resonance spectra were recorded on a Bruker EMX-micro spectrometer equipped with an EMX-Primium bridge and an ER4119HS resonator. Individual solutions were deoxygenated before mixing and the final sample concentrations were 230–250 μM protein, 20–30 μM [Ru(bpy)3]2+, and 4.5 mM [Co(NH3)5Cl]2+. Each sample was ∼100 μL and contained in a flat cell. A dark spectrum was recorded before the sample was exposed to in situ continuous illumination of a 447.5 nm LED (same setup as above) at ambient atmosphere. EPR settings: microwave frequency, 9.85 GHz; microwave power 6.3 mW; modulation frequency 100 kHz; modulation amplitude 0.1 mT. The Xepr software package (Bruker) was used for data acquisition and processing.
9
EPR Measurement of Superoxide Generation
10
Solid-State EPR Spectroelectrochemical Experiment
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The procedure and cell set-up used were as previously reported for solid-state EPR spectroelectrochemical experiments.29,45,46 (link) Briefly, a three electrode set-up consisting of a platinum wire working and auxiliary electrode each with a platinum mesh attached to the ends and a silver wire quasi-reference electrode. The wires were coated with Teflon to prevent short-circuiting with the bottom 1 cm of each wire stripped, with the exception of the auxiliary electrode wire that was completely bare. The sample was immobilised on a platinum mesh connected to the end of the platinum wire working electrode and inserted into a cell consisting of a flame-sealed pipette with the exposed wires well separated. The potential was controlled using an μAutolab II potentiostat with 0.1 M [(n-C4H9)4N]PF6/MeCN used for all measurements. Continuous wave X-band EPR spectra were recorded using a Bruker EMX Micro spectrometer equipped with a 1.0 T electromagnet with the microwave power and attenuation tuned to prevent signal saturation. The EPR signals obtained were referenced to strong pitch to obtain the g-value and spectral simulations were carried out using the Easyspin simulation package.47 (link)
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