X-band EPR spectra were obtained on a Bruker ELEXSYS E-500 X-band spectrometer. The data were collected over a 1500 Gauss field centered at 3100 Gauss. The modulation receiver and signal channel parameters were a modulation frequency of 100 kHz, a modulation amplitude of 10 Gauss, and a time constant of 327 ms. The microwave power was set to 20 mW with a frequency of 9.58 GHz. To determine the number of Ni(III) centers present in each NiSOD variant, spin integration was performed on each EPR spectrum. The resulting EPR spectra were integrated twice and the concentration of Ni in each sample was determined using ICP-OES (λ=231.604 nm). Reduced samples were first treated with 10 molar equivalents of sodium dithionite in an anaerobic environment and analyzed by EPR for the presence(absence) of Ni(III). The reduced samples were buffer exchanged anaerobically with fresh 20 mM Tris, pH 8.5 to remove excess reducing agent and treated with two molar equivalents of IrCl6 to produce the oxidized samples. The oxidized samples were then analyzed by EPR spectroscopy for Ni(III) content.
Elexsys e 500 x band spectrometer
Manufactured by Bruker
The ELEXSYS E-500 X-band spectrometer is a versatile electron paramagnetic resonance (EPR) instrument designed for scientific research. It operates at X-band frequencies and is capable of detecting and analyzing paramagnetic species in various sample types.
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3 protocols using elexsys e 500 x band spectrometer
1
EPR Characterization of Ni(III) in NiSOD
2
X-band EPR Spectroscopy of Frozen Samples
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X-band electron paramagnetic resonance spectroscopy measurements were performed using a Bruker ElexSys E500 X-band spectrometer on the frozen solutions. The EPR measurements were conducted under the following conditions, unless stated otherwise: a modulation frequency of 100 kHz, modulation amplitude of 1 G, microwave power of 2.07 mW and microwave frequency of f ≈ 9.3 GHz. The samples with a volume of 400–500 μL were transferred to the EPR quartz tube sample holder and slowly cooled down to 150 K with a mean cooling rate of ca. 30 K/min. For the fast-freezing experiment, the same procedure as for Mössbauer measurements was applied. The sample geometry with respect to the EPR cavity and the spectrometer settings were kept uniform for all the samples, ensure consistency in the signal amplitude and enable meaningful comparisons of EPR signal intensities. A separate spectrometer background reference was measured by using 500 μL of distilled water (frozen at 150 K), and was subsequently subtracted from the measured spectra prior the further analysis.
3
EPR Analysis of Protein Samples
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EPR experiments were performed on a Bruker ELEXSYS E500 X-band spectrometer equipped with an ER4102ST standard rectangular Bruker EPR cavity fitted to an Oxford Instruments ESR 900 helium flow cryostat. All EPR studies were carried out at 50 K with 4 mW microwave power at 9.479 GHz and 3 mT modulation amplitude at 100 kHz. The presented spectra were averaged from the accumulation of 4 scans to improve the signal to noise ratio. The EPR samples were prepared under anaerobic condition in glove box (Jacomex) and frozen prior to the EPR analysis.
In a first experiment, two 160 µL protein samples were prepared in 50 mM NaOAc pH 5.
In a first experiment, two 160 µL protein samples were prepared in 50 mM NaOAc pH 5.
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