Cdf 426s peltier

CD spectroscopy was performed on a Jasco J-810 spectropolarimeter equipped with a Jasco CDF-426S Peltier set to 25 °C (Easton, MD, USA). Lyophilized peptide was diluted to 0.2 mg/mL in buffer (final concentration was 25 mM phosphate + 100 mM NaF in PBS, pH 7.4), with or without 30% TFE (v/v), placed in a quartz cuvette (1 mm), and after extensive purging with nitrogen, scanned in the region 190–260 nm (scan speed was 20 nm/min). An average of five scans were baseline-subtracted (buffer, 25 mM phosphate + 100 mM NaF in PBS).

Circular dichroism spectroscopy (CD) measurements were performed on a Jasco J-810 spectropolarimeter equipped with a Jasco CDF-426S Peltier set to 25°C. Averages of five scans were baseline-subtracted (PBS buffer; 25 mM phosphate, 150 mM NaCl) and the alpha-helical content was calculated from the molar ellipticity at 222 nm as previously described [16] (link).
For thermal stability experiments, spectra were obtained from 25°C to 80°C with 2.5°C increments. ApoA-I was diluted to 0.2 mg/ml in PBS (final concentration was 25 mM phosphate, 150 mM NaCl, pH 7.4), placed in a 1 mm quartz cuvette and, after extensive purging with nitrogen, scanned in the region 200 to 260 nm (scan speed was 20 nm/min). The Boltzmann function within the GraphPad software (GraphPad Software, Inc., CA, USA) was used to fit the molar ellipticity values at 222 nm of the temperature gradient to a sigmoidal fit curve.

CD measurements were performed using a Jasco J-810 spectropolarimeter equipped with a Jasco CDF-426S Peltier that was set to 25°C. Protein was diluted to 5 μM in 10 mM Tris at pH 7.4 and incubated with or without 200 μg/ml LPS from E. coli or P. aeruginosa, Lipid A from E. coli, LTA from S. aureus, or heparin (200 and 500 μg/ml). Measurements were performed after a 1–5 min or a 30 min incubation at room temperature in a 0.1 cm quartz cuvette. Scans were measured over the far-UV wavelength interval 200–260 nm, with a scan speed of 20 nm/min. An average of five scans for each sample was collected. The baseline (10 mM Tris buffer alone or with different ligands) was subtracted from the spectrum of each sample for normalization.
The α-helical content was calculated according to previously published equations (23 (link)) and reported below: $\%\text{α}-helicalcontent=\frac{\left(\left[\theta \right]222+3000\right)}{\left(-39000+3000\right)}$ Where −3,000 and −39,000 have been established previously as constants based on the helicity of poly-L-lysine as described by (24 (link)): $\left[\Theta \right]Molarellipticity=\frac{\left(\theta 222\right)\times \left(MRW\right)}{\left(10\times c\times l\right)}$ Where θ₂₂₂ = observed ellipticity at 222 nm in millidegrees, c = concentration in g/l, l = path length of the cuvette (cm), and MRW = mean residual weight, that is, molecular weight of the protein (Da)/(number of amino acids). All the experiments were performed at least three times (21 (link), 25 (link)).

All spectroscopic measurements were carried out under ambient conditions. The UV/vis absorption spectra were recorded on a JASCO V-770 or V-670 spectrometer equipped with a PAC-743R Peltier for temperature control. CD spectroscopic measurements were performed with a JASCO J-810 spectropolarimeter equipped with a Jasco CDF-426S Peltier temperature controller or with a customised JASCO CPL-300/J-1500 hybrid spectrometer. CPL spectra were recorded with a customised JASCO CPL-300/J-1500 hybrid spectrometer. Fluorescence spectroscopic measurements were performed on an Edinburgh Instruments FLS981 fluorescence spectrometer. The quantum yields were determined under highly dilute conditions (A <0.05) relative to Oxazine 1 (ϕ_F = 11% in ethanol)^{54 (link)} as a reference compound.

Circular dichroism spectroscopy (CD) measurements were performed on a Jasco J-810 spectropolarimeter equipped with a Jasco CDF-426S Peltier set to 25°C. Averages of five scans were baseline-subtracted (PBS buffer; 25 mM phosphate, 150 mM NaCl) and the alpha-helical content was calculated from the molar ellipticity at 222 nm as previously described [10 (link), 12 (link)].
For thermal stability experiments spectra were obtained from 25°C to 80°C with 2.5°C increments. ApoA-I was diluted to 0.2 mg/ml in PBS (final concentration was 25 mM phosphate, 150 mM NaCl, pH 7.4), placed in a 0.1 mm quartz cuvette and, after extensive purging with nitrogen, scanned in the region 200 to 260 nm (scan speed was 20 nm/min). The Boltzmann function within the GraphPad software (GraphPad Software, Inc., CA, USA) was used to fit the molar ellipticity values at 222 nm of the temperature gradient to a sigmoidal fit curve.
A stopped flow instrument coupled to CD studied the stability of apoA-I proteins in the presence of SDS. Molar ellipticity (222nm) of apoA-I (0.5 mg/ml) was measured after mixing with 100 mmol/l solution of SDS at a volume ratio 1:5. The decrease of protein signal in a sigmoidal shape was recorded and t1/2 values were determined from Boltzmann fit.

All circular dichroism experiments were performed on a Jasco J‐815 spectrophotometer equipped with a JASCO CDF 426S Peltier temperature controller using a quartz cuvette (2 mm path length) at 25°C. The DNA concentration was 10 μM for all the measurements. The scanning range was 220–320 nm with 0.2 nm data pitch, 2 nm bandwidth, and 0.5-sec response. For each sample, 3 accumulations were acquired with a scan speed of 50 nm/min, then blank-corrected with the data from the corresponding buffer without DNA. The subtracted spectra were normalized to molar ellipticity coefficient (Δϵ) according to the following Equation (2): where θ = CD signal in millidegrees, C = DNA concentration in mol/L, and l = path length in cm.

We performed circular dichroism (CD) measurements on a Jasco J-810 spectropolarimeter (Jasco) equipped with a Jasco CDF-426S Peltier set to 25 °C. The peptides were diluted to 5 μm in buffer (Tris, 10 mm, pH 7.4, and MES, pH 5.5) and incubated with 10–300 μg/ml of LPS for 30 min, placed in a 10-mm quartz cuvette and, after extensive purging with nitrogen, scanned over the wavelength interval at 200–260 nm (scan speed: 20 nm/min). We calculated the averages of five scans for each sample. For examination of time dependence, rTCP96 (5 μm) was incubated for 10 and 120 min at 37 °C in the absence or presence of TLR ligands (50 μg/ml) in 10 mm Tris, pH 7.4. The baseline (10 mm Tris, pH 7.4, or 10 mm MES, pH 5.5, buffer, LPS (E. coli), LPS (P. aeruginosa), lipid A (E. coli), LTA (S. aureus), and PGN (S. aureus)) was subtracted from the spectra of each sample (15 (link), 29 (link)).