The oligomeric state of the protein was determined by static- and dynamic light scattering (SLS/DLS). For SLS 50 μM of DDX1 protein was injected on a Superdex 200 10/300 GL column (GE Healthcare) using a Waters FPLC system (Waters Corp.). The Superdex 200 column was connected to a Wyatt Dawn Heleos II multi-angle-light-scattering (MALS) detector (Wyatt Technology) detecting scattered light from 18 different angles. Refractive index, determined after elution from the column and light scattering data were used to calculate the radius of gyration and the corresponding molecular weight with the help of the Astra Software (Wyatt Technology). For DLS, 25 μM of DDX1 protein in a total volume of 60 μl storage buffer were measured in a Viscotek 802 (Malvern Instruments), which records scattered light at fixed 90° angle. Thirty individual traces with four second measurement time each were recorded. All traces with constant intensity were averaged to fit a combined auto-correlation function, which was used to extract the hydrodynamic radii of the particles in solution and the molecular weight was calculated assuming a globular protein shape. All DLS data analysis was performed using the OmniSIZE Software (Malvern Instruments).
Viscotek 802
Manufactured by Malvern Panalytical
The Viscotek 802 is a multi-detector gel permeation chromatography (GPC) system from Malvern Panalytical. It is designed to measure the molecular weight and size distribution of polymers and macromolecules in solution.
Automatically generated - may contain errors
Lab products found in correlation
Omnisize software, by Malvern Panalytical (2 mentions)
Wyatt dawn heleos 2, by Wyatt Technology (1 mentions)
Superdex 200 column, by Wyatt Technology (1 mentions)
Superdex 200 10 300 gl column, by GE Healthcare (1 mentions)
Astra software, by Wyatt Technology (1 mentions)
J 810, by Jasco (1 mentions)
2 protocols using viscotek 802
1
Oligomeric State Determination of DDX1
2
Characterizing SPRY Domain Stability
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The stability of the SPRY domain was characterized by thermal denaturation monitored by a Jasco J-810 circular-dichroism (CD) spectropolarimeter. A solution of 130 µg ml−1 (5 µM) protein in CD buffer (50 mM K2HPO4/KH2PO4 pH 8.0, 250 mM KF, 3 mM DTE) was heated from 293 to 368 K at a rate of 1 K min−1 and unfolding was followed by recording the light polarization at 222 nm. The buffer and wavelength were chosen to optimize the CD signal from the β-sheet structure (Supplementary Fig. S2). Melting curves were fitted to a two-state unfolding equation (Santoro & Bolen, 1988 ▸ ; Fig. 1 ▸ a).
The homogeneity and oligomeric state of the protein were characterized via dynamic light scattering (DLS). 650 µg ml−1 (25 µM) protein in storage buffer was measured in a Viscotek 802 (Malvern Instruments), which records scattered light at a 90° angle. 30 light-fluctuation curves with 4 s measurement time each were recorded. All traces with constant intensity were averaged to fit a combined auto-correlation function, from which the hydrodynamic radius was extracted (Fig. 1 ▸ b). All DLS data analysis was performed using the OmniSIZE software (Malvern Instruments).
The homogeneity and oligomeric state of the protein were characterized via dynamic light scattering (DLS). 650 µg ml−1 (25 µM) protein in storage buffer was measured in a Viscotek 802 (Malvern Instruments), which records scattered light at a 90° angle. 30 light-fluctuation curves with 4 s measurement time each were recorded. All traces with constant intensity were averaged to fit a combined auto-correlation function, from which the hydrodynamic radius was extracted (Fig. 1
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