CD spectroscopy was used to assess the change in pentapeptide secondary structure in solution. Spectra were recorded with a (JASCO PTC 348) spectropolarimeter at 25° C. Peptide solutions of 5 µM were prepared in water and measured using a quartz cuvette with a path length of 1 cm, from 250 to 190 nm. The spectra of each peptide were taken at two different intervals: 0 and 48 h. All the spectra were blank corrected by subtraction of the background experiment.
Ptc 348
Manufactured by Jasco
Sourced in Japan
The PTC-348 is a precision temperature controller designed for laboratory and industrial applications. It features a digital display and intuitive controls for precise temperature regulation. The device can be used with a variety of temperature sensors and heating elements.
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Lab products found in correlation
715 cd spectrophotometer, by Jasco (7 mentions)
715 cd spectropolarimeter, by Jasco (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
J 715, by Jasco (1 mentions)
J 820 spectropolarimeter, by Jasco (1 mentions)
Merck silica gel 60 f254 plates, by Merck Group (1 mentions)
Ammonium formate ar grade, by Merck Group (1 mentions)
Hplc grade, by Merck Group (1 mentions)
V 530 spectrophotometer, by Jasco (1 mentions)
Formic acid, by Merck Group (1 mentions)
Merck silica gel 60, by Merck Group (1 mentions)
Inova spectrometer, by Agilent Technologies (1 mentions)
J 820, by Jasco (1 mentions)
14 protocols using ptc 348
1
CD Spectroscopy of Pentapeptide Structure
2
Characterization of Modified Oligonucleotide Quadruplexes
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CD samples of modified oligonucleotides and their natural counterpart were prepared at ODN concentration of 50 µM by using PBS (Sigma-Aldrich; 10 mM phosphate buffer, 2.7 mM KCl, 137 mM NaCl, pH 7.4) and subjected to the annealing procedure (heating at 90 °C and quickly cooling at 4 °C). CD spectra of all quadruplexes and CD melting/annealing curves were registered on a Jasco 715 CD spectropolarimeter. For the CD spectra, the wavelength was varied from 220 to 320 nm at 100 nm min−1 scan rate, and the spectra recorded with a response of 16 s, at 2.0 nm bandwidth and normalized by subtraction of the background scan with buffer. The temperature was kept constant at 5 °C with a thermoelectrically controlled cell holder (Jasco PTC-348). CD melting/annealing curves were registered as a function of temperature (range: 5–80 °C) for all quadruplexes at their maximum Cotton effect wavelengths. The CD data were recorded in a 0.1 cm pathlength cuvette with a scan rate of 0.5 °C/min.
3
Circular Dichroism Analysis of Quadruplexes
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CD samples of modified oligonucleotides and their natural counterpart were prepared at a ODN concentration of 50 μM by using a potassium phosphate buffer (10 mM KH2PO4/K2HPO4, 70 mM KCl, pH 7.0) and submitted to the annealing procedure (heating at 90°C and slowly cooling at room temperature). CD spectra of all quadruplexes and CD melting/annealing curves were registered on a Jasco 715 CD spectrophotometer by taking the average of three scans. For the CD spectra, the wavelength was varied from 220 to 320 nm at 100 nm min−1 scan rate, and the spectra recorded with a response of 16 s, at 2.0 nm bandwidth and normalized by subtraction of the background scan with buffer. The temperature was kept constant at 20°C with a thermoelectrically controlled cell holder (Jasco PTC-348). CD melting/annealing curves were registered as a function of temperature (range: 20–90°C) for all quadruplexes at their maximum Cotton effect wavelengths. The CD data were recorded in a 0.1 cm pathlength cuvette with a scan rate of 0.5°C/min. The melting temperature (Tm) values provide the best fit of the experimental melting data.
4
Thermal Stability Analysis of LMTK3 Protein
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CD spectroscopy was performed using a Jasco J-715 instrument (Jasco, Tokyo, Japan) equipped with a PTC-348 temperature control unit. Temperature increased from 20 °C to 90 °C at an increment of 1 °C/min, and data points were acquired every 0.2 °C by monitoring a wavelength of 230 nm. For thermal stability experiments, LMTK3 samples of 5.4 µΜ in 200 mM tris buffer (pH 8.0) and 200 mM NaCl were treated with either DMSO 0.4% (v/v) or 8.3 µΜ C36 in DMSO (0.4%) to a total volume of 120 µL in 0.1 cm cuvettes. Data analysis was performed in GraphPad Prism 8.01 software by fitting data in the transition region to a Boltzmann sigmoidal. Apparent Tm values were determined as the point at which the transition was 50% complete.
5
Circular Dichroism Spectroscopy of SPA4 Peptide
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The SPA4 peptide (417 μM) was incubated in 2 mM sodium lactate solution at room temperature for 24 h. The SPA4 peptide then was diluted to 83.4 μM concentration in methanol while maintaining 2 mM concentration of sodium lactate. The spectra were recorded on a Jasco J-715 CD spectropolarimeter with a PTC-348 WI Peltier temperature controller as described earlier.33,34 (link) The samples for CD spectroscopy were maintained at a temperature of 20 °C, and spectra were obtained using a 0.1 cm cuvette pathlength with three accumulations per spectrum. The CD spectra of samples containing SPA4 peptide were corrected by subtracting spectra of solvent mix containing equivalent volume of water in place of the peptide.
6
Circular Dichroism Spectroscopy of G-Quadruplexes
7
Circular Dichroism Analysis of G-Quadruplex
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CD experiments were performed on a JASCO J-820 spectropolarimeter using a 1.0-mm path length cuvette at a total strand concentration of 20 μM. The CD spectra shown in this study are the averages of at least three scans measured from 220 to 350 nm at a scan rate of 50 nm min−1. The temperature of the cell holder was regulated by a JASCO PTC-348 temperature controller, and the cuvette-holding chamber was flushed with a constant stream of dry N2 gas to avoid condensation of water on the cuvette exterior. Before measurement, the sample was heated to 90°C, cooled at a rate of 1°C min−1 and incubated at 0°C for 1 h. Thermal denaturation was measured both in the dilute and liposome conditions at indicated wavelengths. The heating rates were 0.5°C min−1. It is important to note that under all the conditions, denaturation and renaturation profiles of 1cG3 were identical as assessed by measuring CD intensity at 295 nm. This lack of hysteresis and the presence of an isodichroic point confirmed that the transition between single strand and G-quadruplex was two state.
8
Analytical Methods for Natural Product Characterization
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Commercial reagents, all organic solvents and water (HPLC grade), formic acid (95–97%, Laboratory grade), and ammonium formate (AR grade) were purchased from Sigma–Aldrich (Steinheim, Germany).
TLCs were run on Merck silica gel 60 F254 plates (Kenilworth, NJ, USA) and the spots were visualized by means of a UV lamp (Vilber Lourmat VL-4LC, 365 and 254 nm). Silica gel chromatography was performed using Merck silica gel 60 (0.063–0.200 mm). UV experiments were performed on a JASCO V-530 spectrophotometer, equipped with a PTC-348 temperature controller. 1H (500 MHz and 400 MHz) and 13C (125 MHz and 100 MHz). NMR spectra were recorded on an Agilent INOVA spectrometer (Agilent Technology, Cernusco sul Naviglio, Italy) [34 (link),35 (link),36 (link)]; chemical shifts were referenced to the residual solvent signal (CD3OD: δH = 3.31, δC = 49.0 ppm, CDCl3: δH = 7.26, δC = 77.0 ppm). For an accurate measurement of the coupling constants, the one-dimensional 1H NMR spectra were transformed at 64 K points (digital resolution: 0.09 Hz). 1H connectivities were determined by COSY (mixing time 100 ms).
TLCs were run on Merck silica gel 60 F254 plates (Kenilworth, NJ, USA) and the spots were visualized by means of a UV lamp (Vilber Lourmat VL-4LC, 365 and 254 nm). Silica gel chromatography was performed using Merck silica gel 60 (0.063–0.200 mm). UV experiments were performed on a JASCO V-530 spectrophotometer, equipped with a PTC-348 temperature controller. 1H (500 MHz and 400 MHz) and 13C (125 MHz and 100 MHz). NMR spectra were recorded on an Agilent INOVA spectrometer (Agilent Technology, Cernusco sul Naviglio, Italy) [34 (link),35 (link),36 (link)]; chemical shifts were referenced to the residual solvent signal (CD3OD: δH = 3.31, δC = 49.0 ppm, CDCl3: δH = 7.26, δC = 77.0 ppm). For an accurate measurement of the coupling constants, the one-dimensional 1H NMR spectra were transformed at 64 K points (digital resolution: 0.09 Hz). 1H connectivities were determined by COSY (mixing time 100 ms).
9
Circular Dichroism Spectroscopy of DNA Samples
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Circular dichroism (CD) spectra were obtained with a spectropolarimeter (J-820, JASCO Co., Ltd, Hachioji, Japan) in a buffer containing 100 mM KCl, 10 mM K2HPO4 (pH 7.0) and EDTA-2 K at 25 °C. The DNA samples (10 μM) were refolded by cooling from 90 °C to 25 °C at a rate of 0.5 °C min−1. The temperature of the cuvette was controlled by a temperature controller (PTC-348, JASCO Co., Ltd).
10
CD Spectroscopy of G-quadruplex Structures
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CD samples of modified oligonucleotides were prepared at an ODN concentration of 50 μM using a potassium phosphate buffer (10 mM KH2PO4/K2HPO4, 70 mM KCl, pH 7.0) or a sodium phosphate buffer (10 mM NaH2PO4/Na2HPO4, 70 mM NaCl, pH 7.0) and submitted to the annealing procedure: heating at 90°C and slowly cooling at room temperature. CD spectra of all quadruplexes and CD heating-cooling profiles were registered on a Jasco 715 CD spectrophotometer. For the CD spectra, the wavelength was varied from 220 to 320 nm at 100 nm min−1 scan rate, and the spectra recorded with a response of 0.5 s, at 1.0 nm bandwidth and normalized by subtraction of the background scan with buffer. The temperature was kept constant at 5°C with a thermoelectrically-controlled cell holder (Jasco PTC-348). CD heating-cooling profiles were registered as a function of temperature (range: 5–95°C) for all G-quadruplexes at their maximum Cotton effect wavelengths. The CD data were recorded in a 0.1 cm pathlength cuvette with a scan rate of 30°C/h or 10°C/h.
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