CW-EPR spectra were recorded on an EMX-plus (Bruker Biospin GmbH, Germany) X-band EPR spectrometer equipped with a high sensitivity resonator (4119HS-W1, Bruker). The g factor was calibrated in the experimental conditions using the Bruker strong pitch (g = 2.0028). The principal experimental parameters values were microwave frequency of ca. 9.4 GHz, microwave power 0.1 mW, modulation amplitude 5 G, time constant of ca. 80 ms, conversion time of ca. 200 ms. Four scans were accumulated to achieve reasonable signal-to-noise (S/N) ratio, resulting in ca. 20 mins of acquisition time per spectrum. Samples were supplemented by 10% v/v glycerol to ensure homogeneous peptide distributions and avoid water crystallization-induced phase separation. Then, they were introduced into 4 mm outer diameter quartz tubes (Wilmad-Labglass) and freeze-quenched into liquid nitrogen prior to their introduction into the precooled cavity (T = 100 K, achieved by continuous flow liquid nitrogen cryostat). All experimental EPR spectra were analyzed through computer simulation using homemade scripts based on Easyspin toolbox42 (link) environment. Strains on g factor and hyperfine coupling A were used to account for the experimental line broadening.
4119hs w1
Manufactured by Bruker
Sourced in Germany
The 4119HS-W1 is a high-speed, compact laboratory equipment designed for various applications. It features a rotational speed range, adjustable power settings, and a durable build. The core function of this product is to provide a reliable and versatile solution for laboratory tasks requiring high-speed mixing or sample preparation.
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6 protocols using 4119hs w1
1
CW-EPR Spectroscopy of Freeze-Quenched Samples
2
Organic Thin-Film Preparation and ESR Analysis
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Films were prepared under inert
atmosphere by drop-casting 20 μL of solution (concentration
of 5 g/L) onto synthetic quartz glass substrates (Ilmasil PS, QSIL
GmbH) with dimensions of 3 × 25 mm2, followed by annealing
at 150 °C for 120 min after films were dried completely. The
annealed films were placed into synthetic quartz glass tubes (Ilmasil
PS, QSIL GmbH) with 3.8 mm outer and 3.0 mm inner diameters and the
tubes sealed afterward. ESR spectra were recorded at room temperature
on an Elexsys 580 (Bruker Biospin GmbH) spectrometer equipped with
a 4119HS-W1 (Bruker) cavity: microwave frequency, 9.800 GHz; microwave
power, 150 μW (30 dB attenuation, 150 mW source power); modulation
frequency, 100 MHz; modulation amplitude, 0.1 mT.
atmosphere by drop-casting 20 μL of solution (concentration
of 5 g/L) onto synthetic quartz glass substrates (Ilmasil PS, QSIL
GmbH) with dimensions of 3 × 25 mm2, followed by annealing
at 150 °C for 120 min after films were dried completely. The
annealed films were placed into synthetic quartz glass tubes (Ilmasil
PS, QSIL GmbH) with 3.8 mm outer and 3.0 mm inner diameters and the
tubes sealed afterward. ESR spectra were recorded at room temperature
on an Elexsys 580 (Bruker Biospin GmbH) spectrometer equipped with
a 4119HS-W1 (Bruker) cavity: microwave frequency, 9.800 GHz; microwave
power, 150 μW (30 dB attenuation, 150 mW source power); modulation
frequency, 100 MHz; modulation amplitude, 0.1 mT.
3
EPR Spin Scavenging Kinetics Characterization
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EPR spin scavenging experiments
were performed at room temperature (T = 295 ±
1 K) using an EMX-plus (Bruker Biospin GmbH, Germany) X-band EPR spectrometer
equipped with a high sensitivity resonator (4119HS-W1, Bruker). The
g factor was calibrated in the experimental conditions using the Bruker
strong pitch (g = 2.0028). The samples were introduced
into glass capillaries (Hirschmann, 25 μL) sealed at both the
ends and rapidly transferred into the EPR cavity for measurement.
The principal experimental parameters were as follows: a microwave
frequency of ∼9.85 GHz, a microwave power of ∼4.5 mW,
a modulation amplitude of 1 G, a time constant of ∼5 ms, and
a conversion time of ∼12.5 ms. A scan (sweeping time of ∼10
s) was then acquired every 17 s to obtain the kinetics of TEMPOL reduction
over time. All spectra were best simulated and the resulting simulations
were doubly integrated to relatively quantify the concentration of
remaining TEMPOL. Data analysis and simulations based on experimental
data were performed using Xenon software (Bruker Biospin GmbH, Germany)
and lab-made routines based on EasySpin toolbox under MATLAB (Mathworks)
environment.33 (link) The initial decay of TEMPOL
EPR intensity was linearly fitted to estimate the HO• production rate (slope of the fitted curves).
were performed at room temperature (T = 295 ±
1 K) using an EMX-plus (Bruker Biospin GmbH, Germany) X-band EPR spectrometer
equipped with a high sensitivity resonator (4119HS-W1, Bruker). The
g factor was calibrated in the experimental conditions using the Bruker
strong pitch (g = 2.0028). The samples were introduced
into glass capillaries (Hirschmann, 25 μL) sealed at both the
ends and rapidly transferred into the EPR cavity for measurement.
The principal experimental parameters were as follows: a microwave
frequency of ∼9.85 GHz, a microwave power of ∼4.5 mW,
a modulation amplitude of 1 G, a time constant of ∼5 ms, and
a conversion time of ∼12.5 ms. A scan (sweeping time of ∼10
s) was then acquired every 17 s to obtain the kinetics of TEMPOL reduction
over time. All spectra were best simulated and the resulting simulations
were doubly integrated to relatively quantify the concentration of
remaining TEMPOL. Data analysis and simulations based on experimental
data were performed using Xenon software (Bruker Biospin GmbH, Germany)
and lab-made routines based on EasySpin toolbox under MATLAB (Mathworks)
environment.33 (link) The initial decay of TEMPOL
EPR intensity was linearly fitted to estimate the HO• production rate (slope of the fitted curves).
4
EPR Spectroscopy Protocol for RNA Labeling
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Conventional X-band field swept EPR spectra were acquired on an EMX X-band spectrometer (EMXplus from Bruker Biopsin GmbH, Germany), equipped with a high sensitivity resonator (4119HS-W1, Bruker). RNA-labeled sample in 70 mM sodium phosphate buffer at pH 6.5 supplemented by 10% D2O was loaded into glass capillaries (Hirschmann ringcaps, 20 μl) that were sealed at both ends. Standards of known concentrations of PROXYL were used to estimate the labeling efficiency. All experiments were performed at room temperature (295 ± 1 K). Main acquisition parameters were: 0.05 mT amplitude modulation, 1.8 mW incident microwave power, 40 ms time constant, 100 ms conversion time, 1200 points/scan, 15 mT sweep width and at least 3 scans were accumulated per spectrum to achieve reasonable signal to noise (S/N) ratio. Simulations were generated under matlab environment using Easyspin Toolbox (43 (link)).
5
EPR Spin Scavenging Kinetics Quantification
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EPR spin scavenging experiments
were performed at room temperature (T = 295 ±
1 K) using an EMX-plus (Bruker Biospin GmbH, Germany) X-band EPR spectrometer
equipped with a high sensitivity resonator (4119HS-W1, Bruker). The
g factor was calibrated in the experimental conditions using the Bruker
strong pitch (g = 2.0028). Samples were introduced
into glass capillaries (Hirschmann, 25 μL) sealed at both the
ends and rapidly transferred into the EPR cavity for measurement.
The principal experimental parameters were microwave frequency of
∼9.85 GHz, microwave power of ∼4.5 mW, modulation amplitude
of 1 G, time constant of ∼5 ms, and conversion time of ∼12.5
ms. Every 17 s, a single scan (sweeping time of ∼10 s) was
then acquired to obtain the kinetics of TEMPOL reduction over ∼60
min. All spectra were best simulated, and the resulting simulations
were doubly integrated to relatively quantify the concentration of
remaining TEMPOL. Data analysis and simulations based on experimental
data were performed using Xenon (Bruker Biospin GmbH, Germany) and
lab-made routines based on Easyspin Toolbox under Matlab (Mathworks)
environment.22 (link)
were performed at room temperature (T = 295 ±
1 K) using an EMX-plus (Bruker Biospin GmbH, Germany) X-band EPR spectrometer
equipped with a high sensitivity resonator (4119HS-W1, Bruker). The
g factor was calibrated in the experimental conditions using the Bruker
strong pitch (g = 2.0028). Samples were introduced
into glass capillaries (Hirschmann, 25 μL) sealed at both the
ends and rapidly transferred into the EPR cavity for measurement.
The principal experimental parameters were microwave frequency of
∼9.85 GHz, microwave power of ∼4.5 mW, modulation amplitude
of 1 G, time constant of ∼5 ms, and conversion time of ∼12.5
ms. Every 17 s, a single scan (sweeping time of ∼10 s) was
then acquired to obtain the kinetics of TEMPOL reduction over ∼60
min. All spectra were best simulated, and the resulting simulations
were doubly integrated to relatively quantify the concentration of
remaining TEMPOL. Data analysis and simulations based on experimental
data were performed using Xenon (Bruker Biospin GmbH, Germany) and
lab-made routines based on Easyspin Toolbox under Matlab (Mathworks)
environment.22 (link)
6
EPR Spin Scavenging Kinetics Quantification
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EPR spin scavenging experiments were performed at room temperature (T = 295 ± 1 K) using an EMX-plus (Bruker Biospin GmbH, Germany) X-band EPR spectrometer equipped with a high sensitivity resonator (4119HS-W1, Bruker). Samples were introduced into glass capillaries (Hirschmann, 25 µl) sealed at both ends and rapidly transferred into the EPR cavity for measurement. The principal experimental parameters were microwave frequency of ∼9.8 GHz, microwave power of ∼4.5 mW, modulation amplitude 1 G, time constant of ∼5 ms, conversion time of ∼12.5 ms. A scan (sweeping time of ∼10 s) was then acquired every 17 s to obtain the kinetics of TEMPOL reduction over time. All spectra were best simulated and the resulting simulations were doubly integrated to relatively quantify the concentration of remaining TEMPOL [I/I0 = I(t)/I(t = 0)]. Data analysis and simulations based on experimental data were performed using Xenon (Bruker Biospin GmbH) and lab-made routines based on EasySpin Toolbox under Matlab (Mathworks) environment.43 (link)
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