MALS analysis was performed in the National Center for Protein Science Shanghai (NCPSS). 20 μl of 1 mg/ml purified target protein was subjected to SEC-MALS using a WTC-030S5 size-exclusion column (Wyatt, USA) with elution buffer (20 mM Tris–HCl, pH 7.4, 100 mM NaCl) and passed in tandem through a Wyatt DAWN HELEOS II light scattering instrument (Wyatt, USA) and an optilab refractometer (Wyatt, USA). Data collection and analysis were performed with Astra 6 software (Wyatt, USA).
Dawn heleos 2 light scattering instrument
Manufactured by Wyatt Technology
Sourced in United States
The DAWN HELEOS II is a light scattering instrument designed for the determination of molecular weight, size, and interactions of macromolecules and nanoparticles. It utilizes multi-angle light scattering technology to provide precise and accurate measurements.
Automatically generated - may contain errors
Lab products found in correlation
Astra 6, by Wyatt Technology (5 mentions)
Wtc 030s5 size exclusion column, by Wyatt Technology (5 mentions)
Optilab refractometer, by Wyatt Technology (4 mentions)
Astra software, by Wyatt Technology (3 mentions)
T rex refractometer, by Wyatt Technology (2 mentions)
Superdex 200 10 300 gl column, by Agilent Technologies (1 mentions)
Fplc system, by Wyatt Technology (1 mentions)
Superdex 200 10 300 gl column, by GE Healthcare (1 mentions)
Optilab rex refractometer, by Wyatt Technology (1 mentions)
10 protocols using dawn heleos 2 light scattering instrument
1
SEC-MALS for Protein Characterization
2
SEC-MALS Analysis of Flavivirus Proteins
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SEC–MALS experiments were performed using a GE Superdex 200 10/300 GL column–equipped Agilent FPLC system interfaced with a Wyatt DAWN HELEOS II light-scattering instrument, Wyatt T-rEX refractometer, and a Wyatt dynamic light scattering module. The column was pre-equilibrated in 0.2 μm filtered running buffer of 1× PBS + 0.02% w/v sodium azide before each injection. To equilibrate the system at 37 °C, the running buffer was incubated in a water bath and the column was wrapped with THG thermal tape with a digital thermostat regulator (22 ). For DENV1/3/4 and ZIKV Rosetta design variants, 100 μl of 2.5 mg/ml ice-cold protein (250 μg) was injected to the column at a flow rate of 0.5 ml/min in 1× PBS + 0.02% w/v sodium azide. WT proteins produced from stable Chinese hamster ovary DG44 cells were used for SEC–MALS and were injected in varying amounts as previously published (20 (link)): DENV3 at 2.5 mg/ml, DENV4 at 2.47 mg/ml, and ZIKV at 0.97 mg/ml. MALS data were collected and analyzed using Wyatt ASTRA software, version 11.
3
SEC-MALS Protein Characterization Protocol
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SEC-MALS experiments were conducted on an Agilent FPLC system with a Wyatt DAWN HELEOS II light-scattering instrument, superdex 200 10/300 column, Wyatt T-rEX refractometer, and Wyatt dynamic light scattering module. The instruments were equilibrated with SEC buffer 20 mM HEPES pH 7.4,150 mM NaCl, 200 mg/L sodium azide. Protein samples were diluted to 20 uM in SEC buffer. The light scattering was collected at room temperature with a 0.5 ml/min flow rate. MALS data were analyzed using the Wyatt ASTRA software.
4
Biophysical Characterization of Apo-SOD1
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Apo-SOD1 incubated for 1 week under the pH, temperature, concentration,
and ionic strength conditions listed above was analyzed using a DAWN
HELEOS II light scattering instrument (Wyatt Technology), which detects
scattered light at 18 angles with respect to the incident beam. The
light scattering instrument is interfaced to an Agilent FPLC System
with a connected Superdex 200 10/300 GL column (GE Healthcare), a
T-rEX refractometer, and a dynamic light scattering module (Wyatt
Technology). SEC separation and detection of scattered light, absorbance
at 280 nm, and differential refractive index were performed at room
temperature. Data were analyzed, and weight average molar masses as
a function of elution volume were determined using ASTRA software
(Wyatt Technology) with the Zimm fit method, which assumes weak protein–solvent
interactions.12
and ionic strength conditions listed above was analyzed using a DAWN
HELEOS II light scattering instrument (Wyatt Technology), which detects
scattered light at 18 angles with respect to the incident beam. The
light scattering instrument is interfaced to an Agilent FPLC System
with a connected Superdex 200 10/300 GL column (GE Healthcare), a
T-rEX refractometer, and a dynamic light scattering module (Wyatt
Technology). SEC separation and detection of scattered light, absorbance
at 280 nm, and differential refractive index were performed at room
temperature. Data were analyzed, and weight average molar masses as
a function of elution volume were determined using ASTRA software
(Wyatt Technology) with the Zimm fit method, which assumes weak protein–solvent
interactions.12
5
Characterization of Apo-PF-CABD by SEC-MALS
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Apo-PF-CABD (5.2 mg/ml) in 50mM Tris-HCl buffer (pH 7.8) containing 0.5M NaCl and 1mM EDTA was applied at 0.5 mL/min to Superdex 75 gel filtration column in-line with DAWN HELEOS II light scattering instrument (Wyatt Technology) [laser wavelength 658.0nm]. Data collection/analysis used Astra software (Wyatt Technology) version 5.3.4.20. After preparation by overnight dialysis against 50mM Tris-HCl buffer (pH 7.8) containing 0.5M NaCl and either 5mM or 15mM CaCl2, calcium-bound samples of PF-CABD were analyzed similar to apo state.
6
Molecular Weight Estimation of PE8
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MALS analysis was performed to estimate the MW of PE8 in the National Center for Protein Science Shanghai (NCPSS). First, 20 μl of 1.5 mg/ml purified PE8 protein was subjected to SEC-MALS using a WTC-030S5 size-exclusion column (Wyatt, Santa Barbara, CA, USA) with elution buffer (20 mM Tris-HCl, pH 7.4, 100 mM NaCl) and passed in tandem through a Wyatt DAWN HELEOS II light scattering instrument (Wyatt) and an Optilab rEX refractometer (Wyatt). Data collection and analysis were performed with Astra 6 software (Wyatt).
7
SEC-MALS Protein Characterization
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MALS analysis was performed in the National Center for Protein Science Shanghai (NCPSS). 20 μl of 1 mg/ml puri ed target protein was subjected to SEC-MALS using a WTC-030S5 size-exclusion column (Wyatt, USA) with elution buffer (20 mM Tris-HCl, pH 7.4, 100 mM NaCl) and passed in tandem through a Wyatt DAWN HELEOS II light scattering instrument (Wyatt, USA) and an optilab refractometer (Wyatt, USA). Data collection and analysis were performed with Astra 6 software (Wyatt, USA).
8
Structural Characterization of Proteins
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Multiple alignments of amino acid sequences were performed using ClustalX v.2 program [53] . Secondary structure alignment was generated by DSSP v.2.0 [54] and ESpript v.3.0 (http://espript.ibcp.fr/ESPript/ESPript/) [55] . The phylogenetic tree was constructed by the neighborjoining method using MEGA (Molecular Evolutionary Genetics Analysis) v.7.0 software [29] .
Multi-Angle Light Scattering (MALS) Analysis MALS analysis was performed in the National Center for Protein Science Shanghai (NCPSS). 20 μl of 1 mg/ml puri ed target protein was subjected to SEC-MALS using a WTC-030S5 size-exclusion column (Wyatt, USA) with elution buffer (20 mM Tris-HCl, pH 7.4, 100 mM NaCl) and passed in tandem through a Wyatt DAWN HELEOS II light scattering instrument (Wyatt, USA) and an optilab refractometer (Wyatt, USA). Data collection and analysis were performed with Astra 6 software (Wyatt, USA).
Multi-Angle Light Scattering (MALS) Analysis MALS analysis was performed in the National Center for Protein Science Shanghai (NCPSS). 20 μl of 1 mg/ml puri ed target protein was subjected to SEC-MALS using a WTC-030S5 size-exclusion column (Wyatt, USA) with elution buffer (20 mM Tris-HCl, pH 7.4, 100 mM NaCl) and passed in tandem through a Wyatt DAWN HELEOS II light scattering instrument (Wyatt, USA) and an optilab refractometer (Wyatt, USA). Data collection and analysis were performed with Astra 6 software (Wyatt, USA).
9
Characterization of Apo-PF-CABD by SEC-MALS
10
SEC-MALS Protein Characterization
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MALS analysis was performed in the National Center for Protein Science Shanghai (NCPSS). 20 μl of 1 mg/ml puri ed target protein was subjected to SEC-MALS using a WTC-030S5 size-exclusion column (Wyatt, USA) with elution buffer (20 mM Tris-HCl, pH 7.4, 100 mM NaCl) and passed in tandem through a Wyatt DAWN HELEOS II light scattering instrument (Wyatt, USA) and an optilab refractometer (Wyatt, USA). Data collection and analysis were performed with Astra 6 software (Wyatt, USA).
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