The concentrations of reagents in the EPR samples were: 375 μM CblCΔC, 300 μM cob(II)alamin or cob(II)inamide, 500 μM ΔNCblD in Reaction Buffer. The sample containing cob(II)inamide (300 μM) and GSH (4 mM) was prepared in 0.2 M NaOH + 10 % glycerol. All samples were prepared anaerobically. Continuous-wave EPR spectra were collected on a Bruker Elexsys E500 spectrometer using a Super High Q Cavity (Bruker ER 4122SHQE) and Bruker ER4131VT system. A nitrogen-flow cooling system was used. The following experimental conditions were used: modulation frequency, 100 kHz; modulation amplitude, 10 Gauss; microwave power, 2 mW; temperature; 120 K, microwave frequency, 9.45 GHz. Simulations of the EPR spectra were performed by using the EasySpin toolbox run in MATLAB (v. R2015a; Mathworks, Natick, MA)38 (link) . Simulations included variation of the electron g-tensor and electron-nuclear (59Co) hyperfine tensor, with strain parameters introduced for both g- and hyperfine tensor components (see Figure S4 and Table S1 ).
Easyspin toolbox
Manufactured by MathWorks
Sourced in Germany
The EasySpin toolbox is a software package developed for simulating and analyzing electron paramagnetic resonance (EPR) and electron spin resonance (ESR) spectra. It provides a comprehensive set of functions for calculating and fitting EPR spectra, as well as for analyzing experimental data.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using easyspin toolbox
1
Characterization of Cbl Protein Complexes
2
CW EPR Measurements and Simulations
3
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)
4
EPR Spin Scavenging Kinetics Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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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