An Agilent 1200 Series Infinity II HPLC coupled to a DAWN Heleos II multi-angle light scattering detector and Optilab T-rEX refractive index detector (Wyatt Technology) was used for size-exclusion chromatography multi-angle light scattering (SEC-MALS) analysis. PAPP-A, PAPP-A (E483A) samples (100 μL of 1 mg/mL), and PAPP-A2 samples (100 μL of 1 mg/mL, 3 mg/mL, and 6 mg/mL concentrations) were injected onto a Superdex200 Increase 10/300 GL column (Cytiva, Product: 28990944) at 0.5 mL/min for 60 min in PBS. Molecular weights were derived from analysis using Astra 7.0 software (Wyatt Technology) following calibration with BSA.
Optilab t rex refractive
Manufactured by Wyatt Technology
The Optilab T-rEX is a refractive index detector designed for high-performance liquid chromatography (HPLC) applications. It measures the refractive index of the sample as it passes through the flow cell, providing information about the composition and concentration of the analytes. The device features a temperature-controlled flow cell and a precision optical system to ensure accurate and stable refractive index measurements.
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Lab products found in correlation
Superdex 200 increase 10 300 gl column, by Cytiva (1 mentions)
Astra 7, by Wyatt Technology (1 mentions)
Tskgel g3000pwxl cp column, by Tosoh (1 mentions)
Minidawn treos light scattering detector, by Wyatt Technology (1 mentions)
Agilent hplc system, by Agilent Technologies (1 mentions)
Chemstation, by Agilent Technologies (1 mentions)
Ktamicro system, by GE Healthcare (1 mentions)
Astra software, by Wyatt Technology (1 mentions)
4 protocols using optilab t rex refractive
1
SEC-MALS Analysis of PAPP-A Variants
2
Molecular Weight Analysis of PCX
3
Triple-Detection HPLC Analysis of Polymer Samples
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The protocol presented here was developed for an 1100/1200 Agilent HPLC system fitted with a G1365B UV absorbance detector and extended by a miniDAWN TREOS light scattering detector and an Optilab T-rEX refractive index detector, both from Wyatt Technology (see Equipment ). Measurements were controlled through the programs ChemStation and ASTRA V from Agilent Technologies and Wyatt Technology, respectively. Note, however, that PDC composition can be determined with the aid of any setup providing triple-detection by UV absorbance, LS, and RI, and data analysis can be performed with any spreadsheet program [45 (link)] following the procedure for basic LS analysis.
4
SEC-MALS Analysis of IL-17 Complexes
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SEC-MALS experiments were performed at room temperature with an analytical Superdex 200 3.2/300 Increase SEC column, using an Äkta micro system (GE Healthcare) coupled to a miniDAWN TREOS triple-angle light-scattering detector equipped with an Optilab T-rEX refractive index detector (Wyatt Technology). The column was pre-equilibrated with running buffer (20mM Na-phosphate pH 7.4, 150mM NaCl, 0.02% sodium azide). 25–100μg of protein were diluted into 60μL of running buffer and 50μL were run on the SEC column at a flowrate of 0.075mL/min. For the IL-17A binary complex with IL-17RA, a 1.2-fold molar excess of cytokine was used. For the IL-17A ternary complex with IL-17RC and IL-17RA, the cytokine was mixed with a 1.1-fold molar excess of IL-17RC followed by addition of 1.1-fold molar excess of IL-17RA. UV, light scattering and refractive index values were recorded. Peaks of interest were manually selected, and data analysis was carried out with the ASTRA software (version 5.3, Wyatt Technology; RRID:SCR_016255).
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