Selected derivatives were divided into two testing groups, the first of which consisted of derivatives 17e –17i and the second of derivatives 17a –17d . The absorption spectra of the free acridine derivatives and their complexes with ctDNA were measured using a Specord S300 UV-Vis spectrophotometer in a 10 mM Tris-HCl buffer (pH 7.4) at room temperature in a 100-QS quartz cuvette (1 cm path length). The measured UV-Vis data for derivatives 17a –17i were processed graphically using GraphPad Prism 6 software. Binding constants KB were calculated using the Benesi–Hildebrand equation. (Equation (S2), see SM ). Tm measurements were carried out on a Jasco J-810 spectropolarimeter in a 1 mm quartz cuvette 100-QS in a BPES buffer (6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM Na2EDTA, 35 mM NaCl, pH 7.1). Absorption thermal denaturation curves were processed using GraphPad Prism 6, and the results were used to determine the melting temperatures. The CD spectra measurements were conducted using a J-810 Jasco spectropolarimeter in a 100-QS quartz cuvette (1 cm path length) in a 10 mM Tris-HCl buffer (pH 7.4) at room temperature. The results are presented as the mean of at least three independent measurements, and the obtained data were processed using GraphPad Prism 6.
J 810 spectropolarimeter
Manufactured by Jasco
Sourced in Japan, United States, Italy, Germany, United Kingdom, Canada, France
The J-810 spectropolarimeter is a laboratory instrument designed for the measurement of circular dichroism (CD) and other optical rotatory properties of samples. It provides accurate and reliable data on the secondary structure and conformational changes of biological macromolecules such as proteins, nucleic acids, and other chiral compounds.
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Lab products found in correlation
Cdf 426s peltier, by Jasco (11 mentions)
Sephadex lh 20, by Cytiva (7 mentions)
Maxis 4g q tof mass spectrometer, by Bruker (5 mentions)
Ptc 423, by Jasco (5 mentions)
Quartz cuvette, by Hellma (5 mentions)
Spectra manager 2 software, by Jasco (5 mentions)
Ptc 423l, by Jasco (4 mentions)
Pfd 425s temperature controller module, by Jasco (4 mentions)
Avance 2 400, by Bruker (4 mentions)
Avance 3 hd 700, by Bruker (4 mentions)
U2910 ultraviolet spectrophotometer, by Hitachi (4 mentions)
Paclitaxel, by Merck Group (4 mentions)
Suprasil cuvette, by Hellma (4 mentions)
Prism 6, by GraphPad (3 mentions)
V500 spectrometer, by Jasco (3 mentions)
Peltier device, by Jasco (3 mentions)
Ptc 4235, by Jasco (3 mentions)
Suprasil cell, by Hellma (3 mentions)
Sigmaplot 12, by Merck Group (3 mentions)
1 mm quartz cuvette, by Hellma (3 mentions)
841 protocols using j 810 spectropolarimeter
1
Acridine Derivatives Binding Assay
2
Thermal Stability Analysis of Peptides
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Far-UV circular dichroism (CD) spectra of the peptides were recorded at 25°C temperature on a J-810 spectropolarimeter (JASCO International Co. Ltd., Tokyo, Japan). The CD spectra were measured between 260 nm and 185 nm with an optical pathlength of 1 mm, at a peptide concentration of 0.1 mg/mL in Milli-Q water diluted directly from powder. The bandwidth was 2 nm and data pitch 0.5 nm, the scan speed was set to 100 nm/min and the integration time was 1 sec. Ten spectra were accumulated and plotted. The data were analyzed by CDSSTR method [27 ]. To test the thermostability of the peptide structure the CD spectra were recorded between 25 °C and 55 °C temperature with a ramp rate of 5 °C/min on a JASCO J-810 spectropolarimeter by using a Peltier sample holder. The CD spectra were measured immediately between 260 and 185 nm with an optical pathlength of 1 mm, the peptide concentration was 0.1 mg/mL in Milli-Q water. The bandwidth was 2 nm and data pitch 1 nm, the scan speed was set to 100 nm/min, and the integration time was 1 sec. Five spectra were accumulated and plotted.
3
Conformational Analysis of CrataBL Protein
4
Purification and Characterization of NudC Proteins
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E. coli One Shot BL21 Star (DE3) containing the respective plasmid were induced at OD600=0.8 with 1 mM IPTG for 3 h. Purification was performed as described4 (link). All proteins yielded a single band on SDS page (Coomassie staining, Supplementary Fig. 6a ). For preparation of RNA-free pure NudC, 1 M urea was added to His-Trap buffer A and His-Trap buffer B. To ensure dimer formation, all NudC variants were analyzed by size exclusion chromatography on a Superdex 200 10/300 column (50 mM Tris-HCl pH 7.5, 150 mM NaCl, Supplementary Fig. 6b–f ). Folding of all mutants was confirmed by CD spectroscopy (J-810 Jasco spectropolarimeter, 0.2 mg/mL protein in 5 mM Tris-HCl pH 7.5, 5 mM MgSO4, 0.5 mM EDTA and 0.5 mM DTT, 1 nm bandwidth, 0.5 nm data pitch, 20 nm min−1 scanning speed, 1 s response time, Supplementary Fig. 7 ).
5
Far-UV Spectroscopy of HMBS Mutant
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Far‐UV spectra were obtained using a J‐810 Jasco spectropolarimeter (Jasco Europe S.R.L., Cremella, Italia) with a CDF‐426S Peltier element (Jasco) for temperature control and a 300 μL quartz cuvette with a path length of 1 mm. Wt‐HMBS and p.R26H at 5 μm were prepared in 10 mm K2HPO4, 100 mm NaF. Three scans were obtained for each spectrum and the buffer scans were subtracted using the integrated Spectra ManagerTM software. DichroWeb and the CDSSTR analysis method with reference set 3, were used for secondary structure predictions [46 (link), 47 (link)].
6
Circular Dichroism Analysis of Amyloid-β Peptide
7
CD and LD Spectral Analysis of ctDNA-Thio/Urea Interactions
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Spectral measurements of CD were realized using a J-810 Jasco spectropolarimeter in a quartz cuvette with optic length 1 mm. CD spectra of ctDNA (7.46 µM) in the absence or in the presence of the newly developed 7-MEOTA-THA thio-/urea hybrids (12 –22 : 0.3 mM) were recorded in 10 mM Tris–HCl buffer (pH 7.4; RT) in the wavelength range of 200–500 nm. The results are presented as the mean of at least three repeated measurements and obtained data were transferred to Grafit 7.0 (Erithacus Software, West Sussex, UK) for analysis.
Flow LD spectra were measured using a Jasco J-720 spectropolarimeter in a flow Couette cell adapted for LD measurements. The flow cell includes a fixed outer cylinder and a solid rotating quartz inner cylinder. These two cylinders are separated with a 0.5 mm gap (total path length was 1 mm). LD spectra of ctDNA (310 µM) in the absence or in the presence of the newly synthesized 7-MEOTA-THA thio-/urea derivatives (12 –22 : 0–0.1 mM) were recorded in 10 mM Tris–HCl buffer (pH 7.4; RT) in the wavelength range of 200–500 nm. The baseline (10 mM Tris–HCl) was also recorded for each measurement. The results are presented as the mean of at least three independent measurements and obtained data were transferred to Grafit 7.0 (Erithacus Software) for analysis.
Flow LD spectra were measured using a Jasco J-720 spectropolarimeter in a flow Couette cell adapted for LD measurements. The flow cell includes a fixed outer cylinder and a solid rotating quartz inner cylinder. These two cylinders are separated with a 0.5 mm gap (total path length was 1 mm). LD spectra of ctDNA (310 µM) in the absence or in the presence of the newly synthesized 7-MEOTA-THA thio-/urea derivatives (
8
Probing Protein Conformation by CD Spectroscopy
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Circular dichroism (CD) spectra measurements were performed to investigate the effect on protein conformation of DAAO adsorption on nanotubes. CD spectra were recorded using a J-810 Jasco spectropolarimeter (Jasco Co., Cremella, Italy) and analyzed by means of Jasco software [58 (link)]. The far-UV CD spectrum (190–250 nm) of both native (DAAO) and immobilized (f-MWCNTs-DAAO) enzymes was recorded at 15 °C, with a quartz cuvette (cell pathlength of 0.1 cm). All measurements were performed in 50 mM sodium pyrophosphate buffer, pH 7.4, at 0.1 mg/mL protein concentration and corrected for buffer and f-MWCNTs contributions.
9
Purification and Characterization of NudC Proteins
10
Far-UV CD Analysis of Protein Species
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Far-UV CD spectra of the different αS species were acquired in PBS at 20 °C between 200 and 250 nm, using a scan speed of 50 nm min−1 and a bandwidth of 1 nm. Ten accumulations were recorded for each sample, using a 1-mm path length cuvette and a J-810 Jasco spectropolarimeter (Tokyo, Japan), equipped with a thermostated cell holder.
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