All spectra were recorded on a ChirascanTM spectrometer (Applied Photophysics) at 4 °C using 3 μM protein and 4 mm cuvette; 0.5 nm wavelength step and ten repeats at each wavelength. The Tm for each protein was recorded using 3 μM protein; 4 mm cuvette and temperature probe. The temperature range was set to 5–79 °C with steps of 2 °C in between each measurement and 60 sec. setting time. The α-helical unfolding was followed at 222 nm, using ten repeats. The long-term stability, was followed at 37 °C, measuring the CD signal at 222 nm every minute for 8 hours. Prior to all measurement, all samples were dialyzed against 20 mM KPi, 75 mM NaCl, 0.5 mM TCEP, 2% Glycerol, pH 7.3. All CD measurements were recorded in triplicate. The thermal denaturation temperatures were analyzed using the CDpal software45 (link). In the software, data were normalized between 0 and 1, with 1 representing the value of unfolded protein, and the thermal denaturation temperatures were determined by plotting the fraction of α-helical unfolding at 222 nm as a function of temperature and fitting the data to the two-state denaturation model.
Chirascan spectrometer
Manufactured by Applied Photophysics
Sourced in United Kingdom
The Chirascan spectrometer is a high-performance circular dichroism (CD) and absorbance spectrometer designed for the analysis of biomolecular structure and interactions. It provides accurate and reliable measurements of the chiroptical properties of samples, enabling researchers to study the secondary structure and conformational changes of proteins, nucleic acids, and other biological macromolecules.
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Lab products found in correlation
Silica gel, by Qingdao Marine Chemical (6 mentions)
Silica gel, by Qingdao Haiyang Chemical (6 mentions)
Sephadex lh 20, by GE Healthcare (6 mentions)
Sephadex lh 20, by Cytiva (5 mentions)
Xevo g2 q tof mass spectrometer, by Waters Corporation (4 mentions)
Agilent g3250aa, by Agilent Technologies (4 mentions)
P 1020 polarimeter, by Jasco (4 mentions)
Autospec premier p776 spectrometer, by Waters Corporation (4 mentions)
P 2000 polarimeter, by Jasco (3 mentions)
Pro data viewer software, by Applied Photophysics (3 mentions)
Quartz cuvette, by Hellma (3 mentions)
Gf254 plates, by Qingdao Haiyang Chemical (3 mentions)
Gf254, by Qingdao Haiyang Chemical (3 mentions)
Chirascan spectrometer control panel software, by Applied Photophysics (2 mentions)
Avance 800 mhz, by Bruker (2 mentions)
Q tof mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Evolution 300 uv visible spectrophotometer, by Bruker (2 mentions)
Ltq fleet instrument spectrometer, by Thermo Fisher Scientific (2 mentions)
Avance 3 500, by Bruker (2 mentions)
Tensor 27 ftir spectrometer, by Bruker (2 mentions)
181 protocols using chirascan spectrometer
1
Circular Dichroism Analysis of Protein Thermal Stability
2
Circular Dichroism Spectroscopy of ArlR
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Circular dichroism (CD) spectroscopy was performed on a Chirascan TM spectrometer (Applied Photophysics Ltd) with a 0.1 cm path length of the quartz cuvette. The samples were prepared using buffer A (20 mM Tris-HCl, pH 8.0, 150 mM NaCl) and 0.2 mg/mL ArlR. The sample was 200 µL with 1 nm bandwidth length, 185 -260 nm scanning range, and 0.5 s time-per-point. The spectrum of the target protein at 191 -260 nm was obtained over a range of temperatures starting from 20 °C and incrementally increasing to 94 °C, which we used to measure the ArlR melting temperature (Tm). Ultimately, the Tm value was calculated by the Global 3 software, while the secondary structure was visualized and analyzed using the Deconvolution software that comes with the instrument.
3
Circular Dichroism Spectroscopy of Peptides
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Peptides were dissolved in 5 mM Tris amine at a final concentration of between 30 and 50 µM. The pH of the samples was titrated down by adding 0.3% or 1% (v/v) HClO 4 solution in microlitre amounts. ClO 4 -has greater optical transparency than HCl (which has substantial absorbance below 200 nm), hence substitution of HCl by HClO 4 makes a significant improvement on spectral quality in the far-UV region. CD spectra were acquired on a ChirascanTM Spectrometer (Applied Photophysics, Leatherhead, UK) with samples maintained at 310 K. Spectra were recorded from 260 to 180 nm using a 0.5 mm path length and were processed using Chirascan software where a spectrum of the peptide free solution was subtracted and Savitzky-Gorlay smoothing with a convolution width of 5 points applied.
4
Circular Dichroism Analysis of TPMT
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CD measurements were performed as previously described [18] . In short, all spectra were recorded using a Chirascan TM spectrometer (Applied Photophysics, Leatherhead, UK).
Thermal denaturation was determined following the CD signal at 222 nm for α-helical unfolding using 3 µM recombinant TPMT protein in a 4 mm cuvette and a temperature range of 5-79°C. The measurements were repeated three times and performed in the presence of a 10-fold molar excess of the cofactor SAM. The results were processed using the computer software CDpal [20] . A Far-UV CD spectra in the range of 215-260 nm was performed to evaluate global secondary structure of TPMTwt and TPMT*44 (p.Y166C) (data not shown).
Thermal denaturation was determined following the CD signal at 222 nm for α-helical unfolding using 3 µM recombinant TPMT protein in a 4 mm cuvette and a temperature range of 5-79°C. The measurements were repeated three times and performed in the presence of a 10-fold molar excess of the cofactor SAM. The results were processed using the computer software CDpal [20] . A Far-UV CD spectra in the range of 215-260 nm was performed to evaluate global secondary structure of TPMTwt and TPMT*44 (p.Y166C) (data not shown).
5
Comprehensive Analytical Characterization
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Optical rotation was measured on a Jasco P2000 polarimeter. ECD spectrum was measured on a Chirascan spectrometer. NMR spectra were obtained on a Bruker AvanceIII 500 MHz FT-NMR spectrometer. HR-ESI-MS was analysed on an Agilent 6530 Accurate-Mass QTOF. Semi-preparative HPLC was performed on an Agilent 1260 infinity II system including binary pump, autosampler, DAD detector, and YMC J'sphere ODS-H80 (20 × 250 mm, 4 μm) HPLC column. Mobile phase was an isocratic system of methanol/water or acetonitrile/water at flow rate of 3 mL min−1. Thin layer chromatography was performed on pre-coated plates with silica gel or reversed phase C18 (RP-18). Column chromatography was performed using silica gel (40–63 μm) or RP-18 (150 μm) as adsorbents.
6
GdmHCl-Induced Denaturation of AR-LBD
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CD measurement is performed on a Chirascan spectrometer. All CD measurements were performed in buffer (20 mM Tris–HCl, pH 7.5, 200 mM NaCl, 1 mM EDTA, 10 mM 2-ME). For the GdmHCl induced denaturation experiments, 70 μg/ml of AR-LBD WT and mutant proteins with different GdmHCl concentrations were prepared and equilibrated at 25 °C for 1 h. The CD signal was measured at a path of 0.1 cm, and three independent measurement results were averaged.
7
Protein Conformation and Binding Analysis
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Protein conformation was analyzed by CD on a Chirascan spectrometer at 20°C using a 0.5-mm path-length cuvette. Samples were prepared at 1 to 100 μM in 20 mM sodium phosphate (pH 7.5). Protein conformation and binding stoichiometry were assessed by SEC combined with MALS (Wyatt), DLS (Wyatt), and viscometer (Wyatt or Omnisec), using Superdex 200 increase 10/300 or Superose 6 increase (3.2/300 or 10/300) columns, and equilibrated as detailed in Supplementary Methods. Binding affinity was measured by ITC on MicroCal iTC200 or PEAQ-ITC calorimeters, with α-actinin-2 constructs used as titrand at ~20 μM and FATZ-1 constructs or peptides used as ligand at ~200 μM; all prepared in 20 mM sodium phosphate (pH 7.5), 100 mM NaCl, 50 mM arginine, 50 mM glutamic acid, 1 mM β-mercaptoethanol, 1 mM EDTA, and 0.5 mM phenylmethylsulfonyl fluoride. Binding data were processed and fitted using PEAQ-ITC analysis software.
8
Circular Dichroism Analysis of RNA Structures
9
Circular Dichroism Analysis of Mouse cGAS
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Wild type mouse cGAS catalytic domain and its mutants were buffer exchanged into 10 mM phosphate buffer containing 50 mM K2SO4 at pH 7.5. The CD spectra were measured using a Chirascan spectrometer and Chirascan spectrometer Control Panel software (v. 4.5.1848.0) with each protein samples at 3 μM concentration. The spectra from 280 nm to 190 nm wavelength were recorded at room temperature using a 2 mm path length quartz cuvette. The data were processed with Pro-Data Viewer (v. 4.5.1848.0).
10
Circular Dichroism Spectroscopy of Protein Samples
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Protein samples were dialyzed into 10 mM triethanolamine-HCl pH = 7.4, 150 mM sodium fluoride and concentrated to 30 μM. CD spectra (260 to 180 nm, step size of 0.5 nm, bandwidth of 1 nm, dwell-time per data point of 3 s) were recorded on a ChiraScan spectrometer in terms of ellipticity Θ at 20°C in a 1 mm Quartz CD cell (Hellma). Curves of replicate measurements were smoothened using the instrument software. Data points were exported and plotted with the Kaleidagraph software.
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