The drug loading capacity of SFNPs and SF(PF127)NPs were quantitatively analyzed by a UV–vis spectrophotometer (Agilent Cary60, Agilent Technologies Co. Ltd, USA). QR was extracted from the prepared NPs with acetone and measured by the absorbance (374 nm) of supernatant after centrifugation (16,000 g, 10 min). The drug loading (DL) efficiency was measured as follows:
Agilent cary 60
Manufactured by Agilent Technologies
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The Agilent Cary 60 is a UV-Vis spectrophotometer designed for general laboratory use. It provides reliable and accurate absorbance measurements across a wide wavelength range.
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Prism 8, by GraphPad (1 mentions)
Scope a1, by Zeiss (1 mentions)
Live dead baclight bacterial viability kit, by Thermo Fisher Scientific (1 mentions)
Visking mwco 12 000 14 000, by Serva Electrophoresis (1 mentions)
Nano zs zetasizer, by Malvern Panalytical (1 mentions)
Rhb dye, by Merck Group (1 mentions)
Hq40d, by HACH (1 mentions)
Nano z, by Malvern Panalytical (1 mentions)
Model x pert pro, by Malvern Panalytical (1 mentions)
Jsm 7610f, by JEOL (1 mentions)
Jem 1400flash, by JEOL (1 mentions)
Sephadex g 25, by GE Healthcare (1 mentions)
Zetasizer nano ns dts 1060, by Malvern Panalytical (1 mentions)
Uv 2600i uv vis spectrophotometer, by Shimadzu (1 mentions)
Fp 8300 spectrofluorometer, by Jasco (1 mentions)
Alpha spectrophotometer, by Bruker (1 mentions)
T7 express competent cells, by New England Biolabs (1 mentions)
34 protocols using agilent cary 60
1
Quantitative Analysis of Drug Loading Efficiency
2
Spectrophotometric Titration for pKa Determination
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The spectrophotometric titration was used for dissociation constants’ determination, utilizing spectrophotometer Agilent Cary-60 (Agilent Technologies, CA, USA), pH meter WTW InoLab_IDS Multi 9430 (WTW, Czech Republic), and pH electrode SenTix® Mic (WTW, Czech Republic). Data were analyzed by GraphPadPrism 8.0 software (GraphPad Software, CA, USA). The analyzed samples were prepared by adding 20 μL of compound stock solution (2 mg/mL) and 20 μL of 0.1 M hydrochloric acid into the cuvette with ultrapure water (total volume 3.5 mL). The samples were titrated by 0.1 M sodium hydroxide at 20°C while acquiring absorption spectra (190–500 nm) at different pH values. Absorbance at the chosen wavelength was plotted as a function of pH. The inflection point in the given plot was used for pKa value determination.
3
Quantitative Analysis of Adsorption and Ozonation
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After centrifugation, the supernatants of both adsorption solutions and ozonation mixtures were qualitatively analyzed by UV-Vis spectrophotometry in the wavelengths range 190–800 nm in a 1 cm quartz cell. The device used was an Agilent-Cary 60 brand (Agilent Technologies, Santa Clara, CA, USA) equipped with a data processor. Quantitative assessment of both the adsorption and conversion yields was achieved by determining the residual amount of organic compound in the supernatant through liquid phase chromatography coupled with UV detection (HPLC-UV). For this purpose, an Agilent technology model 1200 brand instrument was used under previously optimized conditions (Table S1 ) with a variable wavelength UV detector and a Star analysis software version 6.
4
Analyzing Mg2+ Chelating Properties
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2 mL of methanol solution containing the components in the sample or control group (Table 4 ) was added to a quartz cuvette (BQ-114-2, Chongqing Xinweier Glass Co., Ltd., Chongqing, China). The absorbance at 200–400 nm was scanned by a UV-Vis spectrophotometer (Agilent Cary 60, Agilent Technologies, Santa Clara, CA, USA), and the blank methanol was used for baseline calibration. The absorbance of the control group was subtracted from that of the sample group at the corresponding wavelength to obtain the differential UV-Vis spectra, which were used to analyze the chelating property of the compound with Mg2+.
5
UV Absorbance Measurements of Nitrified Urine
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UV absorbance measurements were done with a UV–Vis spectrophotometer (Agilent Cary 60, Agilent Technologies, Santa Clara, United States) in the range of 200–800 nm. Preliminary tests have shown that in nitrified urine it is difficult to use the wavelength of 254 nm, which is typically used in wastewater as surrogate for organic compounds. Nitrate, which is present in much higher concentrations in nitrified urine (in our case 2080 mg N/L) than in the effluent of WWTPs (about 10 mg N/L), strongly absorbs at wavelengths between 200 and 250 (section 5 and Figure S 3, SI). To prevent any influence of changes in the nitrate concentration on UV measurements (for details see Mašić et al., 2015 (link)), we chose a slightly higher wavelength of 265 nm. For UV–Vis analysis, all samples were diluted by a factor of 10.
6
Spectrophotometric Determination of MDA
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Determination of malondialdehyde (MDA) was carried out using the spectrophotometer Agilent Cary 60 (Agilent, USA). MDA is one of the secondary products of lipid peroxidation. At elevated temperature, a red complex with thiobarbituric acid (TBA) forms in an acidic environment. The absorbance of the resulting color complex MDA-TBA is measured at three wavelengths of 485 nm, 532 nm, and 560 nm.
7
Spectrophotometric Determination of TPI
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The TPI was determined by measuring the absorbance at 280 nm, using an UV-VIS spectrophotometer Agilent Cary-60 (Agilent, Little Falls, DE, USA) [29 ]. Gallic acid was used to generate the calibration curve, and the results were expressed in mg Gallic Acid Equivalent (GAE) per L of sample. The measurements were carried out in triplicate for the calibration curve and for the sample analyses. In the continuous flow analyses, the 280 nm measurements were carried out with a delay of 1 s, so that the sample could be renovated into the flow cell.
8
Characterization of P.gingivalis Adhesion
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P.g (ATCC 33277) was incubated in brain heart infusion (BHI) broth with 5% goat blood at 37 °C for 2 days. Next, bacteria were resuspended in BHI and incubated on titanium plates at 1 × 106 CFU ml−1 for 24 h. Bacterial growth was determined by measuring absorbance at 600 nm using a spectrophotometer (Agilent Cary 60, Agilent Technology, USA). Bacterial survival was determined with fluorescence staining with LIVE/DEAD BacLight bacterial viability kit (L13152, Invitrogen). Briefly, component A and component B were mixed in ddH2O and placed into samples at room temperature in the dark for 15 min. Finally, a fluorescence microscope (Scope A1, Zeiss) was used for observation. The bacterial morphology was observed with SEM (Phenom Pro X, Phenom Scientific).
9
Optimized CRISPR RNA Synthesis
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All DNA strands were ordered from Sangon Biotech Co. Ltd. (Shanghai, China), and RNA strands were ordered separately from Sangon Biotech Co. Ltd. (Shanghai, China) and GenePharma (Shanghai, China). DNA and RNA strands were dissolved in DEPC-treated water and quantified to stock solutions with 100 μM by ultraviolet (UV)–vis absorption at 260 nm with an Agilent Cary 60 spectrophotometer (Agilent Technologies, Australia). All the dsDNA were prepared by hybridization of an equivalent molar amount of the two complementary oligomers at 95°C for 4 min in annealing buffer [20 mM tris-HCl (pH 7.5), 150 mM KCl, 1 mM EDTA, and 50 mM MgCl2], followed by gradient cooling to 25°C at a rate of 0.1°C/s. The cgRNA, scgRNA-1, and scgRNA-2 solutions were also generated by the hybridization of an equivalent molar amount gRNA and the customized iDNA using the same annealing procedure. For the fabrication of scgRNA in CONAN, iDNAs and gRNA were mixed at the molar ratio of 1.2:1 and hybridized using the same annealing procedure. The concentration of scgRNA in CONAN is regarded as the same as the concentration of the used gRNA. NUPACK software was used to model and design all cgRNAs and scgRNAs (36 (link)). The sequences of all oligonucleotides were listed in tables S1 to S4.
10
BOPIM Molecule Synthesis and Characterization
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All chemical molecules used in synthesis and measurement were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Acros (Waltham, MA, USA). 1H NMR and 13C NMR spectral measurements were performed with a Bruker instrument (Billerica, MA, USA) in the Chemistry Department of Middle East Technical University (Ankara, Türkiye). The mass spectra of all molecules synthesized for the first time were determined using a 1200/6210 Accurate-Mass TOF LC/MS (Agilent Technologies, Santa Clara, CA, USA) at the National Nanotechnology Research Center (Bilkent University, Ankara, Türkiye). Agilent Cary 60 and Agilent Cary Eclipse spectrophotometers (Agilent Technologies) were used in our research laboratory for absorption and fluorescence measurements. The Testboy TV335 device (ELBRO AG, Bülach, Switzerland) was used as a digital LED luxmeter to evaluate the intensity of the LED.
The synthesis pathway of the target BOPIM (6 ) is shown in the Scheme .
The synthesis pathway of the target BOPIM (
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