Size exclusion chromatography (SEC) was conducted to evaluate the percentage of soluble IgG aggregations as one of the most important physical instabilities. In order to separate the monomer from aggregated antibody, a 300 mm TSK 3000 SWXL column (Tosoh Biosep,Germany) was employed. Approximately, 20 μL of each sample containing 2.5 mg/mL was injected. The mobile phase consisted of 0.1 M disodium hydrogen phosphate and 0.1 M sodium sulfate (pH 6.8) with flow rate of 0.5 mL/min. The antibody concentration was measured by a UV detector (Waters, USA) at 280 nm. All experiments were performed in triplicate.
Tsk 3000 swxl column
Manufactured by Tosoh
Sourced in Japan, Germany
The TSK 3000 SWXL column is a size exclusion chromatography (SEC) column designed for the separation and analysis of macromolecules such as proteins, polymers, and other high-molecular-weight compounds. It features a wide separation range and is suitable for a variety of applications in analytical and preparative chromatography.
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Lab products found in correlation
Uv detector, by Waters Corporation (1 mentions)
High performance liquid chromatography (hplc), by Shimadzu (1 mentions)
Uv vis diode array detector, by Shimadzu (1 mentions)
Chromolith performance rp 18e column, by Merck Group (1 mentions)
Dawn helleos 2, by Wyatt Technology (1 mentions)
Zetasizer nano s dls, by Malvern Panalytical (1 mentions)
Hplc system, by Shimadzu (1 mentions)
Spd m20a, by Shimadzu (1 mentions)
Dawn heleos 2, by Wyatt Technology (1 mentions)
Optilab rex, by Wyatt Technology (1 mentions)
Uv detector, by Jasco (1 mentions)
6 protocols using tsk 3000 swxl column
1
Evaluating IgG Aggregation via SEC
2
Structural Characterization of DENV2C Proteins
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The tertiary and quaternary structures of the WT and mutant DENV2C proteins were evaluated by gel-filtration chromatography using a TSK3000SWXL column (Tosoh, Japan). The column was coupled to a Shimadzu HPLC and equilibrated using 50 mM sodium phosphate buffer (pH 6.0) containing 200 mM NaCl and 0.05% NaN3 (equilibration buffer). The calibration curve was obtained by using a mixture of the following standard proteins prepared in the equilibration buffer: 1 mg/mL thyroglobulin (Thyr), 1 mg/mL apoferritin (Apo), 50 µg/mL β-amylase (β-amyl), 50 µg/mL bovine serum albumin (BSA), 100 µg/mL ovalbumin (Ova), 100 µg/mL chymotrypsinogen A (Chymo-A), 2 mg/mL aprotinin (Aprot) and 4 µg/mL N-acetyl-L-tryptophan (N-Ac-L-Tryp). DENV2C (WT) and single-point mutants (L50S, L54S, L81N and I88N) of DENV2C were diluted to 30 µM in equilibration buffer. Chromatography was performed at 25 °C with a flow rate of 1 mL/min and an injection volume of 200 µL. Protein elution was monitored by a fluorescence detector with excitation at 280 nm and emission at 340 nm. A calibration curve was obtained by plotting the elution volume of each standard protein versus the logarithm of the molecular weight of the protein.
3
Comprehensive Characterization of Polymer Conjugates
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The synthesis and purity of monomers were monitored by reversed-phase HPLC using Chromolith Performance RP-18e columns (100×4.6 mm, Merck, Germany) with a linear gradient of water-acetonitrile (0-100 % acetonitrile) in the presence of 0.1 % TFA with a UV-VIS diode array detector (Shimadzu, Japan). The molecular mass of the monomers was determined using mass spectrometry performed on an LCQ Fleet mass analyzer with electrospray ionization (ESI-MS) (Thermo Fisher Scientific, Inc., MA, USA). The determinations of the molecular weights and polydispersity of the copolymers and the release of the drug from the polymer conjugate were carried out by size exclusion chromatography (SEC) on a HPLC system (Shimadzu, Japan) equipped with UV, differential refractive index, and multi-angle light scattering (LS) DAWN Helleos II (Wyatt Technology Corp., USA) detectors. For the analysis, a TSK 3000 SWXL column (Tosoh Bioscience, Japan) (80 % methanol, 20 % phosphate buffer pH 6.5) at a flow rate of 0.5 ml/min was used. The content of the thiazolidine-2-thione (TT) groups and cytarabine was determined spectrophotometrically on a Helios Alpha UV/VIS spectrophotometer (Thermospectronic, UK) using the absorption coefficients for TT in methanol (ε 305 =10,800 l•mol -1 •cm -1 ) and for cytarabine in methanol (ε 301 =5,065 l•mol -1 •cm -1 ).
4
Nanoparticle Characterization by DLS and SEC
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Lyophilized nanoparticles were dispersed in PBS (7.4) for one hour at room temperature. Hydrodynamic size and zeta potential measurements were carried out with a Zetasizer Nanos DLS (Malvern Instruments, UK) at further diluted concentrations. The molecular weights and polydispersity of the copolymers were determined by using size exclusion chromatography (Shimadzu HPLC system, Shimadzu) equipped with a refractive index, UV, and multiangle static light scattering (Wyatt Technology Corporation, CA) detectors using a TSK 3000 SWXL column (Tosoh Bioscience, Japan) using 80% methanol, 20% 0.3 M acetate buffer (pH 6.5) at a flow rate of 0.5 mL/min. The calculation of molecular weights from the light-scattering intensity was based on the known injected mass, assuming 100% mass recovery.
5
Molecular Weight Characterization of Polymers
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The number-average molecular weight (Mn), weight-average molecular weight (Mw), and dispersity (Ð) of polymer precursors P1 –P5 and polymer-cluster conjugates POL5 and POL6 were determined by a Shimadzu HPLC system equipped with a size exclusion chromatography (SEC) column TSK 3000 SWXL column (Tosoh Bioscience, Tokyo, Japan). Evaluation was carried out using a multi-angle light scattering (MALS) DAWN HELEOS II (Wyatt Technology Co., Santa Barbara, CA, USA), photodiode array SPD-M20A (Shimadzu, Japan) and differential refractometer index Optilab®-rEX (Wyatt Technology Co., Santa Barbara, CA, USA) detectors. The analysis was performed using a mixture of methanol and 0.3 M sodium acetate buffer, pH 6.5 (4/1, v/v) as a mobile phase at a flow rate of 0.5 mL min−1. The ASTRA software (version 8.1, Wyatt Technology Co., Santa Barbara, CA, USA) was used for calculation of Mw and Ð values.
6
SEC-HPLC Quantification of IgG Aggregates
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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.
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