Attenuated total reflection (ATR) infrared spectroscopy was used to obtain signature spectra for protein and lipid composition of isolated REV samples. 3 µL of fresh REV sample was spotted and dried under ambient conditions on the top of the diamond ATR element of a single reflection Golden Gate accessory (Specac Ltd., Orpington, UK) fitted into a Varian 2000 FT-IR spectrometer (Varian Inc., Paolo Alto, CA, USA). Spectra were recorded by coaddition of 64 individual scans with a nominal spectral resolution of 2 cm−1. Before the spectrum evaluation, ATR correction and the subtraction of the PBS buffer background were performed. For all spectral manipulations, the GRAMS/32 software package (Galactic Inc, Birmingham, AL, USA) were used.
Varian 2000 ftir spectrometer
Manufactured by Agilent Technologies
Sourced in United States
The Varian 2000 FTIR spectrometer is a Fourier transform infrared spectroscopy instrument designed for laboratory use. It is capable of analyzing the infrared absorption spectrum of a sample to identify its molecular composition.
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5 protocols using varian 2000 ftir spectrometer
1
Characterizing REV Composition via ATR-FTIR
2
ATR-FTIR Spectroscopic Protein-Lipid Ratio
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IR spectra were recorded by the means of a Varian 2000 FTIR spectrometer (Varian Inc., US) equipped with a Mercury-Cadmium-Telluride detector and a single reflection diamond ATR accessory (Specac Ltd, UK). A volume of 3 μL of the sample was placed on the top of the diamond ATR crystal and dry film spectra were collected (64 scans, 2 cm−1 spectral resolution) after solvent evaporation. At least three parallel measurements were done. A spectroscopic protein-to-lipid ratio (P/Lspectr) was determined using the protocol described in [12 ]. Briefly, after PBS background spectral subtraction, the relative amount of protein was estimated by the integrated intensity of protein Amide I band (around 1650 cm−1) while the lipid content was approximated by the integrated area of C-H stretching bands from the 3020–2800 cm−1 wavenumber region. Conversion of the spectroscopic protein-to-lipid ratio (P/Lspectr) to nominal protein-to-lipid ratio (P/Lnom) required an adequate calibration curve. For this purpose, BSA–Brain Total Lipid Extract mixtures prepared in PBS buffer with varying protein-to-lipid ratios (from 0.2 to 4 mg/mg) were used.
3
Attenuated Total Reflection FTIR Spectroscopy of CD-Complexes
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IR spectra (128 scans, 4 cm−1 spectral resolution) of the CD-complexes were recorded in attenuated total reflection (ATR) mode using a dynamically aligned Varian 2000 FTIR spectrometer (Varian Inc., Palo Alto, CA, USA) equipped with a liquid nitrogen cooled broadband MCT detector and fitted with a ‘Golden Gate’ single reflection diamond ATR unit (Specac Ltd., Orpington, UK). 5 μL of sample solution was spread on the top of the ATR crystal and a thin dry film was obtained by evaporation of the aqueous solvent under ambient conditions. All spectra were ATR corrected. For spectral manipulations the GRAMS/AI (7.02) software package was used.
4
ATR-FTIR Analysis of Hydrated Lipid Samples
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ATR-FTIR (Attenuated Total Reflection Fourier Transform Infrared) spectra were collected on a Varian 2000 FTIR spectrometer (Varian Inc, USA) using a 'GoldenGate' single reflection diamond ATR accessory (Specac Ltd, UK). 256 scans were acquired at a resolution of 2 cm -1 . Spectra were recorded at room temperature both as hydrated lipid suspension (immediately after pipetting a drop of the sample on the top of the ATR element) and as dry film spectra (after drying the sample on the ATR crystal). ATR correction was applied after each data collection. The spectral deconvolution of selected bands was performed using mixed Gaussian and Lorentzian functions. Band positions were estimated using the second derivative, band shapes were approximated by mixed Gaussian and Lorentzian function until the 2 parameter became minimal. The GRAMS/32 software package was used for all spectral manipulations.
5
FTIR Analysis of Purified EPS
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Functional groups present in the purified EPS were determined by Fourier transform infrared (FTIR) spectroscopy. Samples for infrared analysis were prepared by mixing with spectroscopygrade KBr and prepared in the form of pellets at a pressure of 1 MPa. The pellets were of about 10 mm diameter and 1 mm thickness. The FTIR spectral data were recorded with a Varian 2000 FTIR spectrometer (Varian, Inc., USA). FTIR spectra were recorded covering the 4,000-400 cm -1 region.
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