Far-UV CD was used both for measurement of secondary structure and for validation of the thermostability of TbMORN1 constructs. To avoid the absorption of Tris and NaCl below 180 nm [40 (link)], three protein samples of 100 μl were first dialysed against 2x 1 L of dialysis buffer (20 mM NaH2PO4, 20 mM Na2HPO4, 200 mM NaF) (overnight, 4°C). The pH 8.0 was adjusted by mixing the mono- and dibasic sodium phosphate solutions. The dialysed proteins were clarified by centrifugation in an Optima MAX-XP tabletop ultracentrifuge (Beckman Coulter Life Sciences) (90,720 x g, 30 min, 4°C). The concentration of protein samples was adjusted to 0.25 mg/ml. CD measurements were carried out in a quartz cuvette with an optical path length of 0.5 mm (Stana Scientific Ltd) using a Chirascan Plus spectrophotometer (Applied Photophysics) equipped with the Chirascan-plus DMS software package. The CD profiles for secondary structure calculations were obtained at RT in the range of 190–260 nm. Further analysis was carried out using the BeStSel server, which is specialised in the analysis of CD data from proteins rich in β-strands [41 (link), 42 (link)]. Data were converted to Δε (M-1cm-1) and uploaded to the BeStSel online server. Melting experiments were performed in the range of 190–260 nm, 20–80°C, with a temperature ramp of 0.8°C/min. Data were analysed with Global 3 software package.
Chirascan plus
Manufactured by Applied Photophysics
Sourced in United Kingdom
The Chirascan Plus is a circular dichroism (CD) spectrometer designed and manufactured by Applied Photophysics. It is a versatile instrument capable of measuring the optical activity of chiral molecules over a wide range of wavelengths, from the far-ultraviolet to the near-infrared region. The Chirascan Plus provides high-quality data and advanced features to support research in various fields, including structural biology, biochemistry, and materials science.
Automatically generated - may contain errors
Lab products found in correlation
P 2000 polarimeter, by Jasco (4 mentions)
Nicolet 6700 ft ir spectrometer, by Thermo Fisher Scientific (3 mentions)
Quartz cuvette, by Hellma (2 mentions)
Global 3, by Applied Photophysics (2 mentions)
Cary 100, by Agilent Technologies (2 mentions)
L gsh, by Merck Group (2 mentions)
D gsh, by Koma Biotech (2 mentions)
Avance 800 mhz spectrometer, by Bruker (2 mentions)
Xevo g2 qtof ms spectrometer, by Waters Corporation (2 mentions)
Cdnn software, by Applied Photophysics (2 mentions)
Pro data chirascan 4, by Applied Photophysics (2 mentions)
Peg 200, by Merck Group (1 mentions)
Akta purifier, by GE Healthcare (1 mentions)
Chirascan v100, by Applied Photophysics (1 mentions)
Pro data viewer software, by Applied Photophysics (1 mentions)
7500 real time pcr instrument, by Thermo Fisher Scientific (1 mentions)
Sypro orange protein gel stain, by Merck Group (1 mentions)
Shamrock sr 303i, by Oxford Instruments (1 mentions)
Dv420a oe, by Oxford Instruments (1 mentions)
4 2 hydroxyethyl 1 piperazineethanesulfonic acid, by Merck Group (1 mentions)
144 protocols using chirascan plus
1
Circular Dichroism Analysis of TbMORN1
2
Far-UV CD Spectroscopy of Biomolecules
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Far-ultraviolet (UV) (190–260 nm) circular dichroism (CD) spectra were obtained from the CD spectrometer (ChirascanTM-plus, Applied Photophysics, Ltd., Leatherhead, Surrey, UK) and temperature-regulated cells (25 °C) with 0.5 mm path and 1.0 nm bandwidth. The spectra were taken at pH 4.0, 5.0, and 6.0 in media mentioned above.
3
Far-UV CD Spectroscopy of Biomolecules
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Far-ultraviolet (190–260 nm) CD spectra were obtained from the CD spectrometer (ChirascanTM-plus, Applied Photophysics, Ltd., Leatherhead, Surrey, UK) and temperature-regulated cells (25 °C) with 0.5 mm path and 1.0 nm bandwidth. The spectra were obtained at pH 4–6 in the media mentioned above.
4
Teixobactin Structural Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Teixobactin and compounds 27 and 28 were dissolved in mixed solvent (H2O: MeCN: PBS/60:40:1). The final concentration was 0.5 mg/ml. CD spectrum and data were collected with Circular Dichroism Spectrometer, model Chirascan Plus.
5
Thermal Stability Analysis of D76N-β2m Mutants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Changes in the stability of D76N-β2m as result of insertion of cysteine and MTSL at residues 20, 33, 57, and 88 were evaluated by thermal denaturation using far UV CD (Chirascan Plus). For these experiments, 20 μM of protein, dissolved in 25 mM sodium phosphate, 115 mM NaCl, pH 6.2 was used. The temperature ramp was from 20 °C to 80 °C, at a rate of 1 °C/min. A far UV CD spectrum (195–260 nm) of each protein was acquired at each temperature during the ramp. The data were fitted to two state equilibrium in CDPal software (https://github.com/PINT-NMR/CDpal ) (75 (link)). Where E is the mean residue ellipticity, ΔHm is the change of enthalpy at the midpoint of denaturation (Tm), ΔCp is the change of heat capacity, R the universal gas constant, and T is the temperature (Kelvin).
6
CD Spectroscopy of Oligonucleotides
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD spectroscopy experiments were conducted on a Chirascan Plus spectropolarimeter. Oligonucleotide solutions were prepared at a final concentration of 4 μM in 10 mM lithium cacodylate (pH 7.2) containing 1 mM EDTA supplemented with either 1 mM or 100 mM salt (where the salt is LiCl, NaCl, or KCl). Oligonucleotides were annealed at 95 °C for 3 min and store at 4 °C at least 12 h before analysis. Scans were performed over the range of 200–320 nm at 5 °C. Each trace was the result of the average of three scans taken with a step size of 1 nm, a time per point of 1 s and a bandwidth of 1 nm. A blank sample containing only buffer was treated in the same manner and subtracted from the collected data. The data were finally baseline corrected at 320 nm. Denaturation experiments were performed by heating the samples to 95 °C at a rate of 1 °C min− 1, with data collection every 1 °C. The CD signal at 265 nm was monitored and melting temperature (Tm) values were extracted as the half-maximum decrease in ellipticities.
7
Circular Dichroism of Oligonucleotides
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Oligonucleotides (Supplementary Table S2 ) were diluted to final concentration of 3 μM in 20 mM phosphate buffer, 80 mM KCl over a pH range of 5.4–7.4. Where indicated, PEG200 (Sigma-Aldrich, #88440) was added at 40% (v/v) final concentration. Samples were heated at 95°C for 5 min and then slowly cooled to room temperature overnight. CD spectra were recorded on a Chirascan-Plus equipped with a Peltier temperature controller using a quartz cell with a 5 mm optical-path length. CD data were measured at 20°C from wavelength 230 to 320 nm. Acquired spectra were baseline-corrected for signal contribution from the buffer, and the observed ellipticities were converted to mean residue ellipticity according to θ = degree × cm2 × dmol−1 (molar ellipticity). CD spectra were performed in duplicate and plotted with R.
8
Circular Dichroism of LpxA Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spectra from a Chirascan Plus: steady state Circular Dichroism/Fluorescence spectrometer with titration and automated temperature ramping capabilities were recorded at 25°C using 0.1 cm water-jacketed cell. LpxA samples were diluted to 3–10 μM in 10 mM potassium phosphate, pH 8.0. Spectra were measured from 260 to 190 nm. Molar ellipticity was calculated using Equation 1, where ΔA is the change is CD spectra obtained from 260 to 190 nm, C is the molar concentration of LpxA, and I the cell path length. Following polarimetric conversions molar ellipticity was reported in degrees cm2 dmol−1. Spectra were measured at least three times with independent biological replicates.
9
Evaluating α-synuclein secondary structure
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For evaluation of the secondary structure of α-synuclein protein incubated with CSB, samples were diluted in PBS to 0.5 mg/ml and analyzed using a circular dichroism detector (Chirascan Plus).
10
Purification and Characterization of BfmR Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The E. coli BL21 (DE3) strains carrying pET22b-bfmR were cultured in LB medium at 37 °C until reaching an OD600 of 0.8. Then, 0.4 mM IPTG was added to the cultures for further shaking at 16 °C overnight. The cells were harvested and resuspended in a buffer consisting of 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM Phenylmethanesulfonylfluoride, and 5% glycerol. Ultrasound was used to lyse the cells, and the resulting lysate was centrifuged at 14,000 rpm for 30 min. The supernatant was applied to Ni-agarose resin and eluted with a buffer containing 300 mM imidazole. The obtained proteins were subsequently purified using an AKTA purifier (GE Healthcare) and concentrated to a final concentration of 3 mg/mL. All the mutant proteins were purified using the same method and detected by circular dichroism spectra (Chirascan plus). Briefly, the protein samples were diluted to 0.1 mg/mL in buffer containing 25 mM Tris-HCl pH 8.0, 150 mM NaCl, and placed at 25 °C for 6 h. Then 200 μL sample was taken in a colorimetric dish (1 mm) for testing. The given results were the average of at least 3 parallel detections, followed by background and air deductions.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!