The molar mass/conformation analysis of the polymer solutions were carried out using a size-exclusion chromatography system from Shimadzu (LC20AD pump, CTO-20AC oven and SPD-20A detector), coupled to a DAWN HELEOS II detector (MALS) with an Optilab T-rEX refractometer, both from Wyatt. The solvent used was toluene, and the dn/dc of 0.0592 mL g−1 was determined beforehand. The sampling process was optimised during the in situ polymerisation reaction. The sampled polymer solution was immediately quenched by inserting 300 μL polymer solution sampled in a 10 mL graduated flask containing about 9 mL of toluene, until a total volume of 10 mL was reached. Sampling could be performed solely for 230 s, as beyond that point, a solid gel was formed, in sampling every 10 s, occurring in a window time of 160–230 s.
Optilab t rex refractometer
Manufactured by Wyatt Technology
Sourced in United States, Canada
The Optilab T-rEX refractometer by Wyatt Technology is a precision instrument designed to measure the refractive index of liquids. It provides accurate and reliable data on the refractive index of samples, which is a fundamental property used in a variety of scientific and industrial applications.
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Lab products found in correlation
Astra 6, by Wyatt Technology (13 mentions)
Minidawn treos, by Wyatt Technology (9 mentions)
Astra software, by Wyatt Technology (7 mentions)
Superdex 200 increase 10 300 gl column, by GE Healthcare (6 mentions)
Astra 6.1 program, by Wyatt Technology (5 mentions)
Minidawn treos multi angle light scattering detector, by Wyatt Technology (5 mentions)
658.9 nm laser beam, by Wyatt Technology (4 mentions)
Minidawn treos mals detector, by Wyatt Technology (4 mentions)
Superdex 75 10 300 gl, by GE Healthcare (4 mentions)
Superdex 75 increase 10 300 gl size exclusion column, by GE Healthcare (4 mentions)
Online uv detector, by Shimadzu (4 mentions)
Astra 7, by Wyatt Technology (3 mentions)
Kta pure system, by GE Healthcare (3 mentions)
Superdex 200 10 300 gel filtration column, by GE Healthcare (3 mentions)
Minidawn treos system, by GE Healthcare (3 mentions)
Optilab t rex differential refractometer, by GE Healthcare (3 mentions)
Uv 900 detector, by GE Healthcare (3 mentions)
Astra software package, by Wyatt Technology (2 mentions)
Superdex 200 increase 10 300 or 5 150 column, by Cytiva (2 mentions)
Agilent 1260 infinity 2, by Agilent Technologies (2 mentions)
60 protocols using optilab t rex refractometer
1
In-Situ Polymer Solution Analysis
2
Protein Characterization by SEC-MALS
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The SEC-MALS analysis was performed on a DAWN HELEOS multi-angle light scattering detector, with eighteen angles detectors and a 658.9 nm laser beam, (Wyatt Technology, Santa Barbara, CA, USA) and an Optilab T-rEX refractometer (Wyatt Technology) in-line with two coupled Superdex 200 Increase 10/300 GL size exclusion chromatography analytical columns. Experiments were performed using an isocratic pump (Agilent) with a flow of 0.5 ml/min at room temperature (25°C). Data collection was performed with ASTRA 6.1 software (Wyatt Technology). For the experiments, 300 μl at 1.3 mg/ml protein were loaded on the columns with running buffer of 25 mM HEPES pH 8, 10% glycerol, 300 mM NaCl and 2 mM EDTA. Calibration was performed with high-molecular weight markers from GE Healthcare (thyroglobulin, 669 000 Da; ferritin, 440 000 Da; aldolase, 158 000 Da; conalbumin, 75 000 Da; and ovoalbumin, 43 000 Da).
3
SEC Analysis of Protein Samples
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Experiments were performed on an Agilent 1260 Infinity II HPLC system equipped with a Superdex 200 Increase 10/300 or 5/150 column (Cytiva), a miniDAWN TREOS MALS detector, and an Optilab T-rEX refractometer (Wyatt Technology Corp.). The column was equilibrated in SEC buffer and 40–150 µg of protein were applied after 0.1 µm filtration. Data were processed with the Astra software package (Wyatt Technology Corp.).
4
Protein Molecular Weight Analysis
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All experiments were performed on a Superdex S75 10/300 GL column (GE Healthcare) with a sample injection volume of 250 μL and a flow rate of 0.5 mL/min at 4°C. The volume of the sample loop was 100 μL. Molecular weight markers used to calibrate the column were: albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and ribonuclease A (13.7 kDa) from the Low Molecular Weight Gel-filtration Calibration kit (GE Healthcare). Absolute molecular weight calculations were obtained by static light scattering in line with size exclusion chromatography using the Wyatt Optilab T-rEX refractometer and miniDAWN Treos multiangle light scattering system at 4°C. Protein concentrations were monitored by the refractometer and light scattering unit directly after the gel filtration column. Absolute molecular weights were determined using ASTRA version 6.0 (Wyatt Technologies).
5
Characterization of DUSP26-N (C152S) Protein
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After system equilibration in 50 mM Bis-Tris (pH 6.0), 1 mM DTT at RT, the samples were analyzed with three detectors in series, namely, the UV and light-scattering detectors of the DAWM HELEOS II system (Wyatt Technology) coupled to a refractive-index detector (Optilab T-rEX refractometer; all from Wyatt Technology Corp., Wyatt Technology Europe GmbH, In der Steubach, Germany). As previously described [42 (link)–44 (link)], analysis was performed at RT by injecting the DUSP26-N (C152S) protein sample of 100 μL (2.5 mg/mL) into the SEC-MALS system (WTC-015S5 column, Wyatt Technology) at Korea Basic Science Institute in a mobile phase consisting of 50 mM Bis-Tris (pH 6.0) and 1 mM DTT at a flow rate of 0.5 mL/min. Data were analyzed and weight-averaged molar masses were calculated using ASTRA software (V6, Wyatt Technology).
6
Multi-Angle Light Scattering Analysis
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All MALLS runs were performed using a S200 increase SEC column (10/300 GL, GE Healthcare). Sample injection and buffer flow was controlled by a Hitachi L2130 pump, following the SEC column was a L-2400 UV detector (Hitachi), Optilab T-rEX refractometer (Wyatt technologies) and a DAWN HELEOS-II multi angle light scattering detector (Wyatt technologies). Prior to injection, columns and systems were equilibrated in 5 to 10 column volumes of running buffer. 50 μL injections were performed using protein samples concentrated at a minimum of 2 mg.mL−1, constant flow rate of 0.5 mL.min−1 was used. Accurate MALLS mass prediction was performed with the Astra software (Wyatt Technologies). Curves were represented with Graphpad (Prism).
7
SEC-MALS Analysis of rXKR9 Oligomerization
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SEC-MALS was performed to assess the oligomeric state of rXKR9. Ten micrograms of of purified rXKR9 was filtered through a 0.1 μm filter, injected onto a Superdex 200 Increase 3.2/300 GL column, equilibrated in LMNG SEC buffer, and run at room temperature on an Agilent 1260 Infinity II HPLC coupled with an Eclipse3 system equipped with a miniDAWN TREOS MALS detector and Optilab T-rEX refractometer (Wyatt Technology). Data was analyzed in the ASTRA software package. The dn/dc values used were 0.19 ml/g for rXKR9 and 0.132 ml/g for LMNG.
8
SEC-MALS Analysis of Purified Complexes
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The SEC-MALS system used was an AKTApure FPLC (GE), with an in-line Optilab T-Rex refractometer (Wyatt), and a Dawn Heleos II light scattering instrument (Wyatt). 500 μL of purified complex (~2 mg/mL) was injected onto a WTC-050S5 SEC column (Wyatt) and eluted at 0.5 mL/min directly into the on-line MALS instruments. Data was collected and processed to determine molecular mass using Astra 6 (Wyatt).
9
SEC-MALS Analysis of Immunoglobulin G
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SECMALS analysis was performed by separating approximately 50 μg IgG on a Superdex 200 Increase 10/300 GL column (GE Healthcare Life Sciences) in phosphate buffered saline (140 mM NaCl, 2.7 mM KCl, 5.6 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) and measuring UV absorption at 280 nM and multi-angle light scattering on a DAWN HELEOS II system with Optilab T-rEX refractometer (Wyatt Technology). Raw values were background subtracted and normalized to the maximum signal intensity of each injection. Molecular weights were calculated using the ASTRA6 software package (Wyatt Technology). Data were plotted using GraphPad Prism version 7.0a for Mac (GraphPad Software).
10
Size-exclusion chromatography of purified proteins
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Purified protein samples at a concentration of 0.5 mg ml−1 were injected onto a Superdex Increase 10/300 Gl column (GE Healthcare) that was pre-equilibrated with HBS. The column was coupled in line with a UV-detector (Shimadzu), a miniDAWN TREOS MALLS detector (Wyatt) and an Optilab T-rEX refractometer (Wyatt). Refractive index increment values (dn/dc) of 0.185 ml g−1 and 0.155 ml g−1 were used for protein and glycan analysis, respectively. Bovine serum albumin (Pierce) was used as standard to correct for band broadening. The resulting data was analyzed using the ASTRA6.1 software (Wyatt, v6.1) and errors were calculated in Microsoft Excel.
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