AGT-specific activity was measured at 37 °C, typically using 2.5 (32 nM) to 4 (51 nM, both in subunit) µg·ml−1 purified AGT protein. The reaction mixture contained 10 mM glyoxylate, 100 mM L-alanine, and 150 µM PLP in 100 mM K-phosphate pH 8. The reaction time was set to 2 min and the reaction quenched by using trichloroacetic acid (25% w/v final concentration). Samples were clarified by centrifugation at 21,000 g for 10 min at 4 °C, and supernatants were stored at −20 °C. Pyruvate formed in these reactions was determined using lactate dehydrogenase and NADH 0.2 mM at 37 °C. The reaction was monitored as the change in A340 nm in TRIS-HCl 1 M pH 8 using quartz cuvettes in an Agilent HP8453 spectrophotometer at 25 °C. These amounts of pyruvate were determined using calibration curves of pyruvate under the same conditions. For each variant, we carried out at least five different replicas, and the results were presented as mean ± s.d.
Hp 8453 spectrophotometer
Manufactured by Agilent Technologies
Sourced in United States
The HP 8453 spectrophotometer is a UV-visible absorption spectrophotometer. It measures the absorption of light by a sample as a function of wavelength, providing information about the composition and concentration of the sample.
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7 protocols using hp 8453 spectrophotometer
1
Quantitative Assay of AGT Activity
2
Stability of Mycotoxin Standard Solutions
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UV spectra of individual diluted stock standard solutions of mycotoxins were recorded immediately after their preparation, at 10 and 14 months of storage and utilization. An Agilent HP 8453 spectrophotometer (± 1.0 nm, ±1.0% T) was calibrated before each series of measurements using potassium dichromate solution. Individual mycotoxin standard solution absorption spectrum was taken in triplicate minimum, absorbance at λmax presented in the Table 1 , Table 2 , Table 3 , Table 4 , Table 5 , Table 6 and Table 7 is an average result of parallel measurements. Intra-day variance was calculated for each standard solution. It did not exceed 3% for all studied mycotoxins in solutions besides NIV (4.8%). Thus, mycotoxin concentration was considered constant if variation of average absorbance at λmax within the storage period did not exceed 3%.
3
Photo-uncaging of MRS7145 Monitored by UV-Vis
4
Comparative Analysis of rSol g 4.1 Protein Structures
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Estimations of the secondary structure were performed using CD with a 1 mg/mL solution in a quartz cell cuvette with a path length of 0.5 cm, a scanning rate of 100 nm min− 1 and a range of 190–260 nm on a Jasco J-815 CD Spectrometer (JASCO, Japan) at the Faculty of Science, Khon Kaen University. Excitation and emission spectra were recorded using a slit width of 5 nm, and absorption spectra were measured using an Agilent HP 8453 spectrophotometer. The CD spectra were analyzed to compare the secondary structures of the proteins using Spectra Manager II software. The CD spectra of refolded and non-refolded rSol g 4.1 proteins without the tag were compared. The non-refolded rSol g 4.1 protein was solubilized with 8 M urea and the refolded rSol g 4.1 protein was solubilized in 0.1 mM sodium phosphate buffer, pH 7.4, and CD spectra were recorded.
5
Comprehensive Nanoparticle Characterization
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The sizes of the nanoparticles influence their bioaccumulation and toxicity, and it is of broad importance to perform the characterization of the obtained nanomaterials. The particle diameters, polydispersity indexes, as well as zeta potential measurements were determined by Dynamic Light Scattering (DLS) in a Zetasizer nano-series equipment (Malvern Instruments). The surface plasmon resonance of the nanoparticles was studied using the UV-Vis spectroscopy method with an Agilent HP 8453 spectrophotometer in the wavelength range of 200 to 1000 nm. Furthermore, the morphology and size of silver nanoparticles were evaluated using Scanning Transmission Electron Microscopy (STEM) and Atomic Force Microscopy (AFM) techniques. For DLS, STEM and AFM analyses, the nanoparticles were diluted in MilliQ water 1 : 10 (v/v) and filtered using a 0.22 μm syringe filter. DLS data was acquired using the Zetasizer software, while microscopy analyses were performed using ImageJ.
6
Photouncaging of MRS7344 Compound
7
Infrared, Raman, and UV-Vis Analysis of 4-PPy
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On the Bruker Vertex 80 FT-IR spectrometer, the 4-PPy compound infrared spectra were acquired between 4000 and 550 cm−1. The Raman spectra were recorded using a Bruker RFS 27: Standalone FT-Raman Spectrometer with an InGaAs laser source at 1064 nm in the region 4000–400 cm−1. An Agilent HP 8453 spectrophotometer was used to record the UV–Vis spectrum in a quartz cell using EtOH, MetOH, DMSO, and H2O as the solvent.
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