Absorption spectra were measured using a CARY5000 UV/Vis/NIR spectrophotometer (Agilent). Because the analysed tissues were not transparent, we used an integration sphere in reflectance mode, transforming into absorbance using the Kubelka − Munk function (Nobbs, 1985 ). Resonance Raman spectra were recorded at room temperature and 77 K, the latter with an LN2‐flow cryostat (Air Liquide). Laser excitations at 488, 501.7 and 514.5 nm were obtained with an Ar + Sabre laser (Coherent), and at 577 nm with a Genesis CX STM laser (Coherent). Output laser powers of 10–100 mW were attenuated to <5 mW at the sample. Scattered light was focused into a Jobin‐Yvon U1000 double‐grating spectrometer (1800 grooves/mm gratings) equipped with a red‐sensitive, back‐illuminated, LN2‐cooled CCD camera. Sample stability and integrity were assessed based on the similarity between the first and last Raman spectra.
U1000
Manufactured by Horiba
Sourced in United States
The U1000 is a compact and versatile laboratory equipment designed for a range of analytical applications. It features a high-precision measurement capability and is suitable for use in various research and industrial settings. The core function of the U1000 is to provide accurate and reliable measurement data.
Automatically generated - may contain errors
Lab products found in correlation
Ln2 flowcryostat, by Air Liquide (1 mentions)
Cary 5000 uv vis nir spectrophotometer, by Agilent Technologies (1 mentions)
405 nm diode laser, by Toptica (1 mentions)
Liquid nitrogen cooledccd detector, by Horiba (1 mentions)
Uv 1800 spectrophotometer, by Shimadzu (1 mentions)
Ifs 88 spectrometer, by Bruker (1 mentions)
5 protocols using u1000
1
Spectroscopic Analysis of Biological Tissues
2
Resonance Raman Spectroscopy of DyP Enzymes
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RR and SERR spectra were acquired with a Raman spectrometer (Jobin Yvon U1000, Edison, NJ, USA), equipped with a 1200 lines/mm grating and a liquid-nitrogen-cooled CCD detector, which was coupled to a confocal microscope. An Olympus 20× objective was used for laser focusing onto the sample and light collection in the backscattering geometry. Spectra were measured using a 405 nm diode laser (Toptica Photonics AG, Munich, Germany).
The RR spectra of CboDyP and TfuDyP were measured as previously described [26 (link)]. ScoDyP spectra were acquired at low temperature using ca. 2 μL of the enzyme sample placed in a microscope stage (Linkham THMS 600, Tadworth, UK) cooled to the desired temperature with liquid N2.
RR experiments were performed with a 1.8 mW laser power and a 120 s accumulation time. SERR experiments were performed with a 1.3 mW laser power and 30–40 s accumulation time. Up to 16 spectra were co-added in each measurement to improve the signal-to-noise ratio (S/N). All spectra were subjected to polynomial baseline subtraction; the positions and widths of Raman bands were determined by component analysis as described previously [43 (link)].
The RR spectra of CboDyP and TfuDyP were measured as previously described [26 (link)]. ScoDyP spectra were acquired at low temperature using ca. 2 μL of the enzyme sample placed in a microscope stage (Linkham THMS 600, Tadworth, UK) cooled to the desired temperature with liquid N2.
RR experiments were performed with a 1.8 mW laser power and a 120 s accumulation time. SERR experiments were performed with a 1.3 mW laser power and 30–40 s accumulation time. Up to 16 spectra were co-added in each measurement to improve the signal-to-noise ratio (S/N). All spectra were subjected to polynomial baseline subtraction; the positions and widths of Raman bands were determined by component analysis as described previously [43 (link)].
3
Spectroscopic Analysis of DyP Peroxidases
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UV-Visible and RR experiments were performed using 2–4 μM and 150–300 μM DrDyP or DrDyPM190G, respectively, in 40 mM Britton–Robinson (BR) buffers at different pH values (3–10). UV-Visible spectra were recorded using a Shimadzu UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan) with a temperature-control module set to 18 °C. Formation of DrDyP reaction intermediates was monitored after the addition of excess H2O2 (0.04–4 mM) to the protein solutions. RR solution measurements were completed at room temperature (RT) with 413 nm excitation source. A rotating quartz cell (Hellma, Müllheim, Germany) containing ca. 90 μL of sample was used in all measurements. The spectra were collected in backscattering geometry using a confocal microscope equipped with an Olympus 20× objective (working distance of 21 mm, numeric aperture of 0.35). The microscope was coupled to a Raman spectrometer (Jobin Yvon U1000, Edison, NJ, USA) with a 1200 lines/mm grating and a liquid-nitrogen-cooled CCD detector (Horiba). The laser beam was focused onto the sample with a power of 1.5–3.0 mW and 40–60 s accumulation time. Typically, 4–10 spectra were co-added in each measurement to improve signal-to-noise ratio. RR spectra were subjected to polynomial baseline subtraction and component analysis as described previously [33 (link)].
4
Resonance Raman Spectroscopy of Enzymes
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Resonance Raman (RR) spectroscopic experiments were performed with 2 μL of 0.5 mM enzyme introduced into a liquid-nitrogen-cooled cryostat (Linkam, THMS 600, Tadworth, UK), mounted on a microscope stage, and cooled down to 77 K. Spectra from the frozen samples were collected in backscattering geometry by a confocal microscope coupled to a Raman spectrometer (Jobin Yvon U1000, Edison, NJ, USA) equipped with 1200 1/mm grating and a liquid nitrogen cooled CCD detector. The 413-nm line from a krypton ion laser (Coherent Innova 302, Santa Clara, CA, USA) was used as excitation source. Spectra were accumulated for 60 s with a laser power of 8 mW at the sample with the background scattering removed by subtraction of a polynomial function.
5
Temperature-Dependent Infrared Spectroscopy of FAI
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The infrared spectra of FAI were recorded in a wide temperature range (from 298 to 400 K) in CsI pellets with a Bruker IFS-88 spectrometer in the wavenumber range of 4000–500 cm−1 with a resolution of 1 cm−1 (Fig. S2(b), ESI† ). A SPECAC variable temperature cell (P/N 21.500) was used for high-temperature IR spectra measurements. The Grams/368 Galactic Industries program was used for numerical fitting of the experimental data. Gaussian functions were used for fitting the infrared bands. Powder Raman spectra were measured using a Jobin-Yvon Raman-U1000 spectrophotometer equipped with CCD and photomultiplier (PHT) detectors (Fig. S2(b), ESI† ). The actual spectra were measured with PHT detector applying GAME laser (λ = 532 nm) with power equal to 450 mW for the exciting beam laser. The spectra were measured in the region between 3600 and 10 cm−1 with a resolution of 2 cm−1. The bands characteristic of the internal vibrations of FAI appear in the measured IR spectra. Proposed assignments (see Table S1† ) were determined based on both: (i) theoretical analysis of harmonic IR wavenumbers for formamidine/formamide molecule and cation and (ii) experimental IR and Raman spectra of [FA][Fe(HCOO)3] and [FA][Co(HCOO)3].28 (link)
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