Turbidity was assessed using absorbance readings on a Cary UV-Vis 300 (Agilent), in quartz cuvettes 16.100-Q-10/Z15 (Starna Cells, Inc.). Temperature controlled at room temperature, 20 °C. Absorbance was read at a fixed wavelength of 395 nm for all proteins except sfGFP and NTD-sfGFP, which were read at 600 nm. All samples were assayed in buffers containing 20 mM HEPES, pH 7.5, 1 mM DTT, and other additives as specified.
Cary 300 uv vis
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The Cary 300 UV-Vis is a high-performance spectrophotometer designed for a wide range of applications in analytical chemistry, life sciences, and materials science. It provides accurate and reliable measurements of the absorbance or transmittance of samples in the ultraviolet and visible light spectrum.
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17 protocols using cary 300 uv vis
1
Turbidity Measurement of Proteins
2
Liquid Distribution of Granule Samples
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The liquid distribution (LD) method used is similar to the one reported by Smirani-Khayati et al.
(2009) and has been presented in El Hagrasy and Litster (2013) in detail. Briefly, after completing the sieve analysis, three granule samples from each sieve fraction were dissolved in water separately and sonicated for 1 h. The sonicated samples were further diluted and centrifuged. The supernatant nigrosin dye concentration was measured using a UV/Vis spectrophotometer (Cary UV Vis 300, Agilent, Wilmington, DE) at 𝜆 𝑚𝑎𝑥 = 574𝑛𝑚.
(2009) and has been presented in El Hagrasy and Litster (2013) in detail. Briefly, after completing the sieve analysis, three granule samples from each sieve fraction were dissolved in water separately and sonicated for 1 h. The sonicated samples were further diluted and centrifuged. The supernatant nigrosin dye concentration was measured using a UV/Vis spectrophotometer (Cary UV Vis 300, Agilent, Wilmington, DE) at 𝜆 𝑚𝑎𝑥 = 574𝑛𝑚.
3
Chlorophyll a Extraction from Cell Cultures
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For the extraction of Chlorophyll a from cell cultures, 2 ml of culture were centrifuged for 10’ at 10,000 x g. The supernatant was discarded and 1 ml of DMF (N,N′-dimethylformamide) was added to the pellet. Samples were kept at least for 24 h at 4°C in the dark, to allow the extraction of lipophilic pigments. Pigment concentration was assessed spectroscopically (Cary 300 UV-Vis, Agilent) using the Moran equation (Moran, 1982 (link)).
4
Quantifying InhA Inhibition by Spectrophotometry
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InhA activity assays were monitored by following NADH concentration at 340 nm with a Cary 300 UV-vis (Agilent) spectrophotometer. As described previously [38 (link)], 2-trans-dodecenoyl-CoA (DDCoA) was used as an analogue of the substrate for this reaction. Briefly, to 840 µL of buffer (30 mM PIPES, 150 mM NaCl, pH 6.8) in 1 mL cuvette, inhibitor in DMSO at constant 5% v/v of DMSO (50 µL), 50 µL DDCoA of 1 mM stock in buffer and 50 µL of NADH (Alfa Aesar) of 5 mM stock in buffer were added. After a short period of 2 min pre-incubation at 25 °C, the reaction was started by the addition of 10 µL His6-InhA from a 5 µM stock solution prepared as explained before. Rate of NADH oxidation during His6-InhA activity is monitored in the presence or in the absence of inhibitor. The inhibitory activity of tested molecules is expressed as the percentage inhibition of His6-InhA activity (with respect to control reaction) without inhibitor, as shown below. All activity assays were performed in duplicate or triplicate.
5
Synthesis and Characterization of Copper(II) Pyridine Complex
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Cu(CH3COO)2·H2O (0.1197 g, 0.500 mmol) was dissolved in water (15.00 mL) at room temperature. To the stirred mixture, 2-pyridinemethanol (193 μl, 1.00 mmol) was added. Immediately after adding, the solution change colour to dark blue, and the stirring was continued for next 60 minutes. The resulting blue crystals were obtained after 10 weeks and collected by filtration, washed with CH2Cl2 and diethyl ether. The next step was the process of air drying of the crystals.
C12H12CuN2O2·(4H2O) Yield: 0.0563 g, 0.16 mmol (32% based on Cu(CH3COO)2·H2O).
Anal. calcd for 4 (351.84 g mol−1): C, 40.96; H, 5.73; N, 7.96 [%] found: C, 40.43; H, 5.58; N, 7.80 [%].
Elemental analysis was performed on a FLASH 2000 CHNS Analyzer.
Electronic absorption spectroscopy was carried out with an UV-Vis spectrophotometer (Agilent Technologies, Cary300 UV-Vis).
In order to record the UV-Vis spectra the complexes were dissolved in DMSO/H2O (9 : 1, v/v) and measured in the intervals 0 (after dissolving), 48 and 96 h. The concentration of compounds was 10−6 M to record the whole range, and 10−4 M to record d–d bands, respectively.
C12H12CuN2O2·(4H2O) Yield: 0.0563 g, 0.16 mmol (32% based on Cu(CH3COO)2·H2O).
Anal. calcd for 4 (351.84 g mol−1): C, 40.96; H, 5.73; N, 7.96 [%] found: C, 40.43; H, 5.58; N, 7.80 [%].
Elemental analysis was performed on a FLASH 2000 CHNS Analyzer.
Electronic absorption spectroscopy was carried out with an UV-Vis spectrophotometer (Agilent Technologies, Cary300 UV-Vis).
In order to record the UV-Vis spectra the complexes were dissolved in DMSO/H2O (9 : 1, v/v) and measured in the intervals 0 (after dissolving), 48 and 96 h. The concentration of compounds was 10−6 M to record the whole range, and 10−4 M to record d–d bands, respectively.
6
Multimodal Characterization of Nanomaterials
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The hydrodynamic size and optical properties were measured in Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) and Cary 300 UV-Vis (Agilent, Santa Clara, CA, USA), respectively. The photothermal effect was induced with an 808-nm CW laser (CNI laser, Changchun, China). A confocal laser scanning microscope (Leica TCS SP8, Leica Microsystems GmbH, Wetzlar, Germany) and a microplate reader (VERSAmaxTM, Molecular Devices Corp., Sunnyvale, CA, USA) were used to observe the in vitro cell imaging test. The fluorescence recovery by enzymatic probe cleavage was observed by using the iBrightTM Imaging System (Invitrogen by Thermo Fisher Scientific, MA, USA). The fluorescence and temperature were measured in IVIS Lumina (Caliper LifeSciences, Waltham, MA, USA) and an infrared thermal imaging camera (FLIR, Wilsonville, OR, USA), respectively. The centrifuge was performed using Avanti J-E Series (Beckman Coulter, IN, USA).
7
Determining Thermal Stability of H-24 and H-20
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Samples containing 0.5 mM of H-24 or H-20 in the presence or absence of ssDNA1 were prepared in 37 mM citric acid and 126 mM Na2HPO4 (pH 6) buffer, or 100 mM Tris (pH 9) buffer. The Tt was obtained by measuring the absorbance of the samples at 380 nm as a function of temperature and pH in a temperature-controlled UV–vis spectrophotometer (Cary 300 UV − vis, Agilent) as previously described46 (link). The Tt was obtained by taking the maximum in the first derivative of the absorbance as a function of temperature of triplicate samples.
8
Determining ELP Fusion Protein T_t
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To determine the Tt of the ELP fusion proteins, the optical turbidity at 350 nm of a 25 μM solution of the ELP fusion proteins was measured at a thermal ramp rate of 1°C/min between 4° and 60°C on a temperature-controlled ultraviolet-visible (UV-vis) spectrophotometer (Cary 300 UV-Vis, Agilent Technologies). The LCST phase transition is indicated by the sudden increase in optical turbidity, and the inflection point of the OD350 v. temperature curve was used to calculate the Tt.
9
Inhibitory Effect of Neem Extracts on Trypanosome Alternative Oxidase
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The recombinant trypanosome alternative oxidase (rTAO) used in this study was kindly provided by Dr. Emmanuel O. Balogun, Department of Biomedical Chemistry, Graduate School of Medicine, University of Tokyo, Japan. Inhibitory effect of the PPFs and crude methanol extract of Azadirachta indica was carried out on TAO using the ubiquinol-1 oxidase assay [24 (link)]. Briefly, 10 μg of each fraction/extract (sample) was pre-incubated with 75 ng rTAO and 50 mM Tris–HCl, pH7.4 for 2 min in a 1 mL quartz cuvette. Thereafter, 150 µM of ubiquinol-1 was added to initiate the reaction. A pre-incubated rTAO, Tris–HCl and ubiquinol-1 only served as control. The inhibitory activity of the samples on the enzyme was measured by recording change in absorbance at 278 nm with UV–Vis spectrophotometer (Cary 300 UV–Vis Agilent Technologies).
10
Chlorophyll and Carotenoid Quantification
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To evaluate the chlorophyll a and total carotenoid content, 2 mL of culture were centrifuged for 10 min at 17500 g. The supernatant was discarded, and the pellet was solubilized in 1 mL of DMF (N,N′-dimethylformamide). Samples were kept at 4°C in the dark for at least 24 h, to allow the extraction of lipophilic pigments. Pigment spectra were recorded with a spectrophotometer (Agilent Cary 300 UV-Vis) by using optical glass cuvettes. The concentrations of chlorophyll a and total carotenoids were calculated using the Moran equations for DMF (Moran, 1982 (link)).
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