EPR spectra were recorded at room temperature using an X-band spectrometer at 9.8 GHz employing 100 kHz field modulation. 5,5-Dimethyl-pyrroline N-oxide (DMPO) (400mM, 200μL) was added to cell pellets resuspended in 200μL phosphate-buffered saline solution (pH 7.4). Suspended pellets were either treated with 1mM H2O2 or 50 mM ascorbate. Measurements were taken using a small volume TE102 variable temperature aqueous cell EPR tube (Wilmad-LabGlass, Vineland, NJ). The EPR spectra were recorded on a X-band Bruker EMX 6/1 spectrometer (Bruker Biospin Corp., Billerica, MA) before and after adding 44μL of 1 mM H2O2. The microwave of the EPR was set at a frequency of 9.8 GHz and the power at 6.9 mW. Measurement conditions were as follows: scan width, 100 G; resolution, 1024; receiver gain, 1 χ 104; conversion time, 10.24 ms; time constant, 0.64 ms; number of scans, 8.
Emx 6 1 spectrometer
Manufactured by Bruker
The EMX 6/1 spectrometer is a compact and versatile electron paramagnetic resonance (EPR) spectrometer designed for research and routine applications. It offers a frequency range of 9.5 GHz and is capable of operating at room temperature. The spectrometer provides a stable and reliable platform for conducting EPR measurements.
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5 protocols using emx 6 1 spectrometer
1
EPR Spectroscopy of DMPO-Trapped Radicals
2
Characterization of Complex I
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Protein concentration was determined according to the biuret method using BSA as a standard78 (link). The concentration of purified complex I was determined by the difference of absorbance at 280–310 nm (TIDAS II, J&M Aalen) using an ε of 781 mM−1 cm−1 as derived from the amino acid sequence79 (link). SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) was performed according to Schägger80 (link) with a 10% separating gel and a 3.9% stacking gel. EPR spectra were recorded at 40 and 13 K with a Bruker EMX 6/1 spectrometer operating at X-band33 (link). The samples were reduced by an addition of 1.000-fold molar excess NADH and a few grains of dithionite. Mass photometry was measured with a OneMP (Refeyn) as described48 (link),49 (link). One µL of a concentrated sample (1 µM) of the preparations of complex I and the D213GH variant were diluted with A*MNG buffer to 50 nM on the cover slip and after autofocus stabilization, movies of 60 s duration were recorded. Data were acquired with AcquireMP (Refeyn Ltd., v.1.2.1).
3
EPR Spectroscopy of Solid Samples
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The EPR measurements were carried out at room temperature with a Bruker EMX-6/1 spectrometer in X-band with a cylindrical cavity of type 4119HS W1/0430 using the following acquisition parameters: 350.5 mT central magnetic field; 9.88 GHz microwave frequency; 32 mW microwave power; 100 kHz modulation frequency; 0.5 mT modulation amplitude; 163.84 ms time constant and 81.92 sweep time, 5 averaged scans per one spectrum. The 150–180 mg samples were positioned in the central region of the EPR cavity in a quartz EPR tube of 4 mm inner diameter. Each sample was measured at three orientations of the sample tube in the cavity and the spectra were averaged. Intracavity standard sample (Mn2+ in MgO) was measured simultaneously with all samples and can be seen as two sharp lines at the spectra wings in the presented signals (Figures 3 , 5 , 6A ).
4
EPR Analysis of Complex I Redox States
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EPR measurements were conducted with an EMX 6/1 spectrometer (Bruker) operating at X-band. The sample temperature was controlled with an ESR-9 helium flow cryostat (Oxford Instruments). Spectra were recorded at 40 K and 2 mW microwave power and at 13 K and 5 mW microwave power from 300 to 380 mT. Other EPR conditions were: microwave frequency, 9.360 GHz; modulation amplitude, 0.6 mT; time constant, 0.164 s; scan rate, 17.9 mT min−1. 300 µL complex I (2.5–3.5 mg mL−1) in buffer A were reduced with a 2000 fold molar excess NADH (10–14 mM) and shock frozen at 150 K in 2-methylbutane/methylcyclohexane (1:5; v:v).
To determine whether H2O2 oxidizes or damages cluster N1b, complex I was incubated with 1 mM H2O2 for 5 min. The excess H2O2 was removed by concentrating the sample by ultrafiltration (Amicon Ultra-15, MWCO: 100 kDa, Millipore; 3800 g, 4 °C, rotor A-4-44, centrifuge 5804R, Eppendorf) and subsequent tenfold dilution in buffer A with 5 mM MgCl2, 10% (v/v) glycerol and 0.005% (w/v) LMNG. This procedure was repeated two times. An EPR spectrum of the concentrated sample reduced by a 2000 fold molar excess NADH was recorded.
To determine whether H2O2 oxidizes or damages cluster N1b, complex I was incubated with 1 mM H2O2 for 5 min. The excess H2O2 was removed by concentrating the sample by ultrafiltration (Amicon Ultra-15, MWCO: 100 kDa, Millipore; 3800 g, 4 °C, rotor A-4-44, centrifuge 5804R, Eppendorf) and subsequent tenfold dilution in buffer A with 5 mM MgCl2, 10% (v/v) glycerol and 0.005% (w/v) LMNG. This procedure was repeated two times. An EPR spectrum of the concentrated sample reduced by a 2000 fold molar excess NADH was recorded.
5
Spectroscopic Characterization of Organic Compounds
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All reagents and solvents were acquired commercially and usually used without further purification. The solvents used during the reactions were dried and distilled using typical methods. Analytical TLC was performed on Merck Kieselgel GF 254, 0.2 mm plates supported on aluminium with the described eluent for each case.
NMR spectra were acquired with Bruker ARX 400 or Bruker Avance III 400 spectrometers at NOVA School of Science and Technology. 1H NMR and 13C NMR spectra were measured at 400 and 101 MHz, respectively. The samples were prepared in 5 or 3 mm NMR tubes using CDCl3 or DMSO‐d6 and the corresponding trace as reference signals. The NMR signals are described with chemical shift (δ, in ppm), source of signal (R−H) and relative intensity of signal multiplicity (nH, with n being the number of protons) of NMR signals are described as singlet, broad singlet (br s), doublet of doublets (dd), triplet of doublets (td), doublet (d), triplet (t) and multiplet (m) with the coupling constant (J) being given in Hz. X‐band EPR spectra were acquired with a Bruker EMX 6/1 spectrometer and an ER 4102ST cavity (Bruker), at 298 K and with a modulation frequency of 100 kHz, modulation amplitude of 0.05 mT and microwave power of 635 μW.
NMR spectra were acquired with Bruker ARX 400 or Bruker Avance III 400 spectrometers at NOVA School of Science and Technology. 1H NMR and 13C NMR spectra were measured at 400 and 101 MHz, respectively. The samples were prepared in 5 or 3 mm NMR tubes using CDCl3 or DMSO‐d6 and the corresponding trace as reference signals. The NMR signals are described with chemical shift (δ, in ppm), source of signal (R−H) and relative intensity of signal multiplicity (nH, with n being the number of protons) of NMR signals are described as singlet, broad singlet (br s), doublet of doublets (dd), triplet of doublets (td), doublet (d), triplet (t) and multiplet (m) with the coupling constant (J) being given in Hz. X‐band EPR spectra were acquired with a Bruker EMX 6/1 spectrometer and an ER 4102ST cavity (Bruker), at 298 K and with a modulation frequency of 100 kHz, modulation amplitude of 0.05 mT and microwave power of 635 μW.
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