Polymer tube samples were analyzed before and after e-beam sterilization. The PEU tube material was dissolved in tetrahydrofuran and filtered through a 0.2 μm PTFE syringe filter before analysis. Gel permeation chromatography (GPC) (Wyatt Technology, Santa Barbara, CA) was then used to measure the molecular weight of the tubing. The GPC system consisted of an Agilent 1200 HPLC (Agilent, Santa Clara, CA) with refractive index (Optilab T-rEX, Wyatt Technology, Santa Barbara, CA) and a multi-angle light scattering detector (DAWN HELEOS-II 8 angle, Wyatt Technology, Santa Barbara, CA), and the data were processed with ASTRA software (Version 7.0). The separation columns used were a PLgel guard column (10 μm, 7.5 × 50 mm) and GPC column PLgel Mixed-B column (10 μm, 7.5 × 300 mm) at 25°C. The flow rate of the mobile phase, tetrahydrofuran, was 0.7 ml/min, and the injection volume was 100 μl.
Dawn heleos 2 8 angle
Manufactured by Wyatt Technology
Sourced in France
The Dawn HELEOS-II 8-angle is a multi-angle light scattering (MALS) instrument. It measures the intensity of scattered light at multiple angles to determine the molar mass and size of macromolecules in solution.
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Lab products found in correlation
Astra 6, by Wyatt Technology (3 mentions)
Agilent 1200 hplc, by Agilent Technologies (1 mentions)
Optilab t rex, by Wyatt Technology (1 mentions)
Agilent 1260 infinity lc chromatographic system, by Agilent Technologies (1 mentions)
Superdex 200 increase 5 150, by GE Healthcare (1 mentions)
Optilab t rex refractive index detector, by Wyatt Technology (1 mentions)
Superdex 200 hr 10 30 column, by GE Healthcare (1 mentions)
Optilab rex, by Wyatt Technology (1 mentions)
Sil 20a, by Shimadzu (1 mentions)
Optilab rex, by Shimadzu (1 mentions)
Spd 20a, by Shimadzu (1 mentions)
4 protocols using dawn heleos 2 8 angle
1
Molecular Weight Analysis of Polymer Tubes Before and After E-beam Sterilization
2
Size-Exclusion Chromatography of Mycobacterial Proteins
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Samples of Mtb-SecBTA at 49.1 µM (1.0 mg ml−1), Mtb-SecBTA/HigA1 at 6.5 µM (0.75 mg ml−1), and Mtb-HigA1ΔC42 at 100 µM (1.4 mg ml−1) in 20 mM MES, 200 mM NaCl, pH 6.5 were analyzed on a Superdex 200 Increase 5/150 (GE Healthcare) with multi-angle light scattering. The column was equilibrated with the same buffer, previously sterilized on a 0.1-µm filter, on an Agilent 1260 Infinity LC chromatographic system (Agilent Technology). Data were collected on a DAWN HELEOS-II 8-angle and Optilab T-rEX refractive index detector (Wyatt Technology, Toulouse, France). We loaded 75–100 μl of each protein sample and separation was performed at a flow rate of 0.4 ml min−1 at 15 °C. Results were analyzed with ASTRA 6.0.2.9 software (Wyatt Technology Corp.).
3
Size-Exclusion Chromatography of Protein Complex
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OAZ-GSY-SPM at 2 mg/mL in 100 μL 50 mM HEPES, 150 mM NaCl, 2 mM TCEP, 0.02 mM PLP, pH 7.4 was injected at 25 °C into a Superdex 200 HR 10/30 column (GE Healthcare), connected to a Shimadzu HPLC system with a Wyatt Dawn HELEOS-II 8-angle light scattering detector and Wyatt Optilab rEX refractive index monitor. The running buffer was HBS with 2 mM TCEP and 0.02 mM PLP. ASTRA 6 software (Wyatt Technology) was used for analysis of the light scattering, refractive index, and UV absorbance data. Error bars in the observed molecular weight represent the uncertainty in fitting to the molar mass curve.
4
SEC-MALS analysis of RGMB-GDF5-ActR2b complex
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A SEC-MALS experiment was performed using a Wyatt Dawn HELEOS-II 8-angle light-scattering detector (with 663.8-nm laser) and a Wyatt Optilab rEX refractive index monitor linked to a Shimadzu HPLC system comprising LC-20AD pump, SIL-20A autosampler and SPD20A UV/Vis detector. SEC-MALS of the RGMBND–GDF5–ActR2bECD complex (1 mg mL−1, 100 μL per injection) was performed using Superdex 200 HR 10/30 column equilibrated in 0.5 M NaCl, 20 mM Hepes pH 7.4, 0.5 mL min−1 flow rate, 21 °C. GDF5 variant Tyr487Lys/Gln489Asp with increased affinity for the type 2 receptor was used to reconstitute the RGMBND–GDF5–ActR2bECD complex. SEC-MALS of the RGMBND–GDF5 complex (1 mg mL−1, 100 μL per injection) and RGMBND (1 mg mL−1, 100 μL per injection) were performed using a Superdex 200 HR 10/30 column equilibrated either in 0.5 M NaCl, 20 mM Hepes pH 7.5, or 0.5 M NaCl, 20 mM MES pH 5.5 at 0.5 mL min−1 flow rate, 21 °C. Scattering data were analyzed and molecular weight was calculated using ASTRA 6 software (Wyatt Technology).
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