SEC analyses were carried out using a Superdex 200 Increase 10/300 GL column connected to an AKTA Pure system (GE Heatlhcare) and equilibrated with buffer 75 mM HEPES pH 7.5, 250 mM NaCl and 5 mM MgCl2. Samples containing 200 µg of protein were loaded into the column and were eluted isocratically at a flow rate of 1 mg/ml. Peaks were collected and checked by SDS-PAGE. Chromatograms were exported and analysed in GraphPad Prism software. In the SEC with multi-angle light scattering (SEC-MALS) experiments the chromatographic system was coupled to a Wyatt DAWN HELEOS-II MALS instrument and a Wyatt Optilab rEX differential refractometer (Wyatt). The Astra 7.1.2 software from the manufacturer was used for acquisition and analysis of the data.
Dawn heleos 2 mals instrument
Manufactured by Wyatt Technology
Sourced in United States
The DAWN HELEOS-II MALS instrument is a multi-angle light scattering (MALS) system designed for the characterization of macromolecules and nanoparticles. The core function of this instrument is to measure the absolute molar mass and size of samples in solution by detecting the intensity of light scattered at multiple angles.
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Lab products found in correlation
Astra 6, by Wyatt Technology (3 mentions)
Wyatt optilab rex differential refractometer, by Wyatt Technology (2 mentions)
Optilab rex differential refractometer, by Wyatt Technology (2 mentions)
Kta fast protein liquid chromatography system, by GE Healthcare (2 mentions)
Astra 7, by Wyatt Technology (1 mentions)
Superdex 200 increase 10 300 gl, by GE Healthcare (1 mentions)
Superdex 200 increase 10 300 gl gel filtration column, by GE Healthcare (1 mentions)
Optilab t rex differential refractometer, by Wyatt Technology (1 mentions)
Superdex 200 10 300 gl column, by GE Healthcare (1 mentions)
Agilent 1200, by Agilent Technologies (1 mentions)
Agilent 1200 hplc system, by Agilent Technologies (1 mentions)
Astra 5 software, by Wyatt Technology (1 mentions)
7 protocols using dawn heleos 2 mals instrument
1
Size Exclusion Chromatography Analysis
2
SEC-MALS Analysis of Protein Oligomers
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SEC-MALS experiments were performed using a Wyatt DAWN HELEOS-II MALS instrument and a Wyatt Optilab rEX differential refractometer (Wyatt) coupled to an AKTA pure system (GE Heralthcare)35 . 50 μl of protein at 5 mg ml−1 were injected in a KW-803 (Shodex) column equilibrated with 25 mM Tris pH 8, 250 mM NaCl. When testing the peptide in the AimRKat oligomeric state, 1 mM peptide was added to the injected sample and the running buffer was supplemented with peptide at a final concentration of 1 μM. The Astra 7.1.2 software from the manufacturer (Wyatt) was used for acquisition and analysis of the data.
3
Oligomeric State Analysis by SEC-MALS
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SEC-MALS experiments were performed using an AKTA pure system (GE Heralthcare) coupled to a Wyatt DAWN HELEOS-II MALS instrument and a Wyatt Optilab rEX differential refractometer (Wyatt). 50 μL of the protein samples were injected at a concentration of 5 mg/mL on a Superdex 200 HR 10/300 (GE Heralthcare) column equilibrated in 25 mM Tris pH 8, 250 mM NaCl. When the effect of the peptide in the oligomeric state was tested, 1 mM peptide was added to the injected sample and the running buffer was supplemented with peptide at a final concentration of 1 μM. The Astra 7.1.2 software from the manufacturer was used for acquisition and analysis of the data.
4
SEC-MALS Analysis of SdgB and SdgA
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The oligomeric states of the recombinant SdgB and SdgA proteins were assessed by SEC-MALS experiments using an ÄKTA fast protein liquid-chromatography (FPLC) system (GE Healthcare) connected to a Wyatt DAWN HELEOS II MALS instrument and a Wyatt Optilab T-rEX differential refractometer (Wyatt, Santa Barbara, California, USA). A Superdex 200 Increase 10/300 GL (GE Healthcare) gel-filtration column pre-equilibrated with a buffer consisting of 20 mM Tris–HCl pH 7.5, 200 mM NaCl or a buffer consisting of 20 mM Tris–HCl pH 8.5, 200 mM NaCl for SdgB or SdgA, respectively, was normalized using ovalbumin. The SdgB (0.22 mg) or SdgA (0.23 mg) proteins were injected at a flow rate of 0.5 ml min−1. The data were analyzed using the Zimm model for fitting static light-scattering data and graphs were otained using EASI Graph (Easy Analytic Software Inc.) with an ultraviolet (UV) peak in the ASTRA 6 software (Wyatt).
5
Oligomeric State Determination of MINERVA Proteins
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The oligomeric states of the recombinant MINERVAFL and MINERVAΔC proteins were assessed by SEC–MALS experiments using an ÄKTA fast protein liquid chromatography (FPLC) system (GE Healthcare, Chicago, IL, USA) connected to a Wyatt DAWN Heleos II MALS instrument and a Wyatt Optilab T-rEX differential refractometer. A Superdex 200 Increase 10/300 GL gel-filtration column (GE Healthcare, Chicago, IL, USA), which was pre-equilibrated with buffer A containing 20 mM Tris-HCl (pH 8.0) and 200 mM NaCl, was normalized using ovalbumin. The proteins (2 mg) were injected at a flow rate of 0.5 mL min−1. Data were analyzed using the Zimm model for fitting static light-scattering data and were graphed using Easy Analytic Software Inc. (EASI) graph with a UV peak in the ASTRA 6 software (Wyatt, Santa Barbara, CA, USA).
6
SEC-MALS Analysis of Vps4E233Q
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SEC–MALS experiments were performed using an Agilent 1200 high-performance liquid chromatography system (Agilent Technologies, Santa Clara, CA, USA) coupled to a Wyatt DAWN HELEOS-II MALS instrument and a Wyatt Optilab rEX differential refractometer (Wyatt, Santa Barbara, CA, USA). For chromatographic separation, 200 μl of 5 mg ml−1 (50 μM) Vps4E233Q protein preincubated with 1 mM ATP at 4 °C for 1 h was injected onto a Superdex 200 10/300 GL column (GE Healthcare) at a flow rate of 0.3 ml min−1 in the assembly buffer. The outputs were analysed by the ASTRA 6.0 software (Wyatt). MALS signals, combined with the protein concentration determined by the refractive index, were used to calculate the molecular mass.
7
Structural Analysis of AP-1:Arf1:Tetherin-Nef Complex
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