IgG-containing samples were analyzed for quantification of aggregates/fragments by SEC-HPLC. A TSK 3000 SWXL column (Tosoh, Biosep, Germany) was applied. The running buffer was composed of 0.1 M Disodium hydrogen phosphate dihydrate and 0.1 M Sodium sulfate, and pH 6.8. The flow rate of pump (Jasco, USA) was set to 0.5 mL/min and the injection volume to 20 μL. Each weighed sample containing 2.5 mg of IgG was dissolved in 1 ml of deionized water and mixed. Then the solution was filtered through 0.45 μ syringe filters before analysis. Antibody aggregates, monomers, and fragments were determined using a UV detector (Jasco, USA) at 280 nm. For each sample, analysis was performed in triplicate.
Uv detector
Manufactured by Jasco
Sourced in Japan, United States
The UV detector is a laboratory instrument used to detect and measure the presence of compounds that absorb ultraviolet (UV) radiation. It is designed to provide quantitative analysis of samples by measuring the absorption of UV light at specific wavelengths.
Automatically generated - may contain errors
Lab products found in correlation
High performance liquid chromatography (hplc), by Jasco (3 mentions)
Tsk 3000 swxl column, by Tosoh (1 mentions)
High pressure pumps, by Jasco (1 mentions)
Guard column, by Bio-Rad (1 mentions)
Intelligent autosampler, by Jasco (1 mentions)
Chromatographic oven, by Shimadzu (1 mentions)
Hitrap, by Cytiva (1 mentions)
Ysp 101, by YMC (1 mentions)
Fused silica capillary tube, by GL Sciences (1 mentions)
7 protocols using uv detector
1
SEC-HPLC Quantification of IgG Aggregates
2
Quantification of CRM and NGF Release
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To a 3.3 30 cm2 membrane tube in a flask with 20 mL of DPBS at pH 7.4, 1 mg/mL of LIP carriers in 2 mL of DPBS containing 0.05% sodium azide at pH 7.4 was added and shaken in a bath-reciprocal shaker at 50 rpm and 37°C for 2 days. Then, 100 µL of the sample containing CRM or NGF was treated with 1% (v/v) Triton-X-100 at 4°C for 1 h. The volume of the medium was compensated with 100 µL of fresh DPBS when sampling. To evaluate the quantity of released CRM, the sample was treated with methanol at 37°C for 30 min, analyzed using a HPLC (Jasco) with a reverse-phase BDS Hypersil C-18 column (Thermo Hypersil-Keystone, Bellefonte, PA, USA) at 37°C, and detected using an UV detector (Jasco) at 430 nm. The mobile phase used ultra-pure water (Barnstead) containing citric acid at pH 3 with a tetrahydrofuran gradient from 5% to 40% (v/v), driven using 2 high pressure pumps (Jasco) at a flow rate of 1 mL/min for 20 min. To assess the release of NGF, the sample was analyzed with a human NGF kit using an ELISA spectrofluorometer at 450 nm.
3
Quantification of Allicin Degradation Kinetics
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Aqueous 20 mM CSA and allicin solutions: (1.5 mL) each were transferred to a reaction vessel and incubated at 100 °C in a heating block. A sample was taken every 10 min, and the relative amount of substance was quantified by high-pressure liquid chromatography (HPLC). Separation was performed using H2O as mobile phase A and methanol as mobile phase B with the following gradient: 56% A (pre-run); 53% A (10 min); 7% A (15 min); 7% A (30 min); 56% A (31 min); 56% A (35 min) at a flow rate of 1 mL/min on a C18-reverse phase column. The detection was performed with a UV detector (Jasco GmbH, Groß Umstadt, Germany) at 254 nm.
4
HPLC Analysis of Furan Compounds
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The concentrations of 5‐HMF, FFA, FFCA and FDCA were determined using HPLC (JASCO, Tokyo, Japan) equipped with a fast acid analysis chromatographic column connected to a guard column (Biorad, Richmond, CA, USA), refractive index detector (ERC, Kawaguchi, Japan), a JASCO UV detector operating at 215 nm and a JASCO intelligent autosampler. The column temperature was maintained at 65°C in a chromatographic oven (Shimadzu, Tokyo, Japan). Samples were diluted with Milli‐Q quality water and mixed with 20% (v/v) H2SO4 (20 µl ml−1 sample) and then filtered. A 40 µl aliquot was injected in 0.5 mM H2SO4 mobile phase flowing at a rate of 0.4 ml min−1. The peaks for the different compounds were confirmed and quantified using the corresponding external standards.
To determine the FAD bound to the enzyme, 500 µl of 1 mg ml−1 of pure MycspAAO‐WT was filtered using 30 kDa cut‐off and washed twice using 500 µl of 0.1 M phosphate buffer pH 8, and then denatured at 100°C for 15 min and separated by centrifugation at 15 000 g for 5 min. The released FAD was detected at 450 nm in a spectrophotometer (Boateng et al., 2015 (link), Dishisha et al., 2019 (link)), and the concentration was calculated based on a standard curve.
To determine the FAD bound to the enzyme, 500 µl of 1 mg ml−1 of pure MycspAAO‐WT was filtered using 30 kDa cut‐off and washed twice using 500 µl of 0.1 M phosphate buffer pH 8, and then denatured at 100°C for 15 min and separated by centrifugation at 15 000 g for 5 min. The released FAD was detected at 450 nm in a spectrophotometer (Boateng et al., 2015 (link), Dishisha et al., 2019 (link)), and the concentration was calculated based on a standard curve.
5
Peptide Stability Analysis by HPLC
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For the examination of the chemical stability, the peptide was analyzed with high-performance liquid chromatography (HPLC, JASCO, Tokyo, Japan) using a cation-exchange column (HiTrap-SP, Cytiva). TPN-RQ 33.0 μM, which was mixed with an equal volume of PBS or mouse serum, was analyzed with HPLC immediately after mixing or after incubation at 37°C for 30 min. Peptides were eluted with a gradient of NaCl from 200 to 2,000 mM in 10 mM Tris-HCl (pH 7.4), monitoring A230 nm with a UV detector (JASCO).
6
Capillary LC System for Separation
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In this work, all experiments were conducted by using a capillary LC system constructed by a syringe pump YSP-101 (YMC, Kyoto, Japan) equipped with a gas-tight syringe (0.5 mL; Ito, Fuji, Japan) as a pump, an C4-1004-.2 internal sample injector (VICI Valco Instruments, Houston, USA) with an injection volume of 0.2 μL, a microcolumn prepared from a fused-silica capillary tube (100 × 0.32 mm i.d.; GL Sciences), a UV detector (JASCO, Tokyo, Japan) with the detection wavelength set at 210 nm, and a data processor (CDS-Lite Ver. 5.0; LA soft, Chiba, Japan). The inlet pressure was monitored by an L.TEX-8150 pressure sensor (L.TEX Corporation, Tokyo, Japan). Separation columns were operated under room temperature (controlled at 25°C) throughout the study.
7
Capillary LC System for Separation
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In this work, all experiments were conducted by using a capillary LC system constructed by a microfeeder (L.TEX Corporation, Tokyo, Japan) equipped with a gas-tight syringe (0.5 mL; Ito, Fuji, Japan) as a pump, an M-435 micro valve injector (Upchurch Scientific, Oak Harbor, WA, USA) with an injection volume of 0.2 μL, a microcolumn prepared from a fused-silica capillary tube (100 × 0.32 mm i.d.; GL Sciences, Tokyo, Japan), a UV detector (JASCO, Tokyo, Japan) with a wavelength 210 nm, and a data processor (CDS-Lite Ver. 5.0; LA soft, Chiba, Japan). The inlet pressure was monitored by an L.TEX-8150 Pressure Sensor (L.TEX). Separation columns were immersed into a water bath for temperature controlled 20°C throughout the study.
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