To directly measure the molecular mass of SARS-CoV-2 Ubl1 in solution, the size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) assay was performed. The purified 50 μL Ubl1 (~5 mg/mL) protein was injected into column WTC-030S5 (Wyatt Technology, USA) with a flow rate of 0.75 mL/min. The light scattering signal and the refractive index profile were collected with the DAWN and Optilab (Wyatt Technology, USA). The molecular mass was calculated by the software ASTRA 8 (Wyatt Technology, USA).
Astra 8
Manufactured by Wyatt Technology
Sourced in United States, Germany
The ASTRA 8 software is a comprehensive data analysis suite designed for researchers and scientists. It provides a powerful platform for the analysis and interpretation of data generated by various laboratory instruments, including light scattering, chromatography, and spectroscopy systems. The software offers a wide range of analytical tools and features to support the characterization of macromolecules, colloids, and other complex materials.
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Lab products found in correlation
Superdex 75 increase 10 300 gl column, by Cytiva (2 mentions)
Optilab refractometer, by Wyatt Technology (2 mentions)
Superdex 200 increase 10 300, by GE Healthcare (2 mentions)
Wtc 030s5, by Wyatt Technology (1 mentions)
Superose 6 10 300 gl, by Cytiva (1 mentions)
Superdex 200 10 300 gl, by Cytiva (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Pierce bovine serum albumin standard ampules, by Thermo Fisher Scientific (1 mentions)
Minidawn mals detector, by Wyatt Technology (1 mentions)
Optilab differential refractive index detector, by Wyatt Technology (1 mentions)
Agilent 1260 infinity 2, by Agilent Technologies (1 mentions)
Optilab refractive index detector, by Wyatt Technology (1 mentions)
Minidawn light scattering detector, by Wyatt Technology (1 mentions)
Superdex 200 increase column 10 300 gl, by Wyatt Technology (1 mentions)
Minidawn 3 angle light scattering detector, by Wyatt Technology (1 mentions)
Superdex 75 increase 10 300 gl column, by GE Healthcare (1 mentions)
Optilab t rex refractive index detector, by Wyatt Technology (1 mentions)
Superdex 200 10 300 increase column, by Agilent Technologies (1 mentions)
Multi wavelength absorbance detector, by Wyatt Technology (1 mentions)
Heleos 2 8 multi angle light scattering detector, by Wyatt Technology (1 mentions)
17 protocols using astra 8
1
Measuring SARS-CoV-2 Ubl1 Mass by SEC-MALS
2
Characterization of Recombinant SKR-1 Proteins
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Recombinant SKR-1 proteins (0.1 mg/ml) and SYP/SKR-1 complexes (0.1 mg/ml) were separated on Superdex 200 10/300 GL and Superose 6 10/300 GL columns (Cytiva), respectively, that were equilibrated with 10 mM Hepes, 400 mM NaCl, 0.5 mM EGTA, 5 mM MgCl2, 2 mM tris(2-carboxyethyl)phosphine (TCEP), and 5% glycerol at a flow rate of 0.1 ml/min. The light scattering and reflective index profiles of recombinant SKR-1 proteins were collected by miniDAWN and OptiLab (Wyatt Technology). Data were collected every second and analyzed by ASTRA 8 (Wyatt Technology). Bovine serum albumin (Sigma-Aldrich, P0834) was used to calibrate the light scattering detector.
3
SEC-MALS Analysis of LmPC-PLC Molar Mass
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Molar mass characteristics of the LmPC-PLC were determined using SEC connected to a UV as concentration detector operating at a wavelength of 280 nm (Agilent Technologies, USA) and a DAWN multi-angle light scattering photometer (Wyatt Technology Corp., USA). Separations were performed at room temperature using a Tricorn 10/300 Superdex 75 GL SEC column (10 mm ID × 30.0 cm L, 8.6 µm particle size, 70 kDa exclusion limit, Sigma-Aldrich, USA). Buffer (500 mM NaCl and 20 mM Tris-HCl pH 8.5) with a flow rate of 0.5 ml/min was used as mobile phase. Toluene was used to calibrate the 90° LS detector, while the other detectors were normalized using Bovine Serum Albumin (BSA) as an isotropic scatterer (Pierce™ Bovine Serum Albumin Standard Ampules, ThermoFisher, USA). The injection volume was 100 μL. The specific refractive index increment (dn/dc) used to calculate the molar mass of LmPC-PLC was 0.185 ml/g. Astra 8.0.2.5 software (Wyatt Technology Corp., USA) was used for data acquisition and analysis.
4
SEC-MALS Analysis of Protein Samples
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For SEC‐MALS measurements, a Superdex 75 Increase 10/300 GL column (Cytiva Life Sciences) coupled to an ÄKTA pure™ protein purification system was used, which was further connected to a miniDAWN MALS detector and an Optilab differential refractive index (dRI) detector (Wyatt Technology). Measurements were performed at room temperature using buffer C with the addition of 0.02% sodium azide, 0.8 mL min−1 flow rate, and protein concentrations of 1, 2, and 3 mg mL−1 with an injection volume of 100 μL. Reproducibility of the SEC‐MALS runs was ensured by obtaining identical results in the measured BSA (bovine serum albumin) samples (Pierce) at 2 mg mL−1 as the first and last sample of each day's measurements. The first BSA injection was used for detector normalization, peak alignment, and band broadening. The dRI signal was used as a concentration source for molar mass determination in all cases. Data were collected and analyzed using ASTRA 8.0.2.5 software (Wyatt Technology).
5
SEC-MALS Characterization of Proteins
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SEC‐MALS measurements were performed using a Superdex 75 Increase 10/300 GL column (Cytiva Life Sciences) connected to an Agilent 1260 Infinity II HPLC system, coupled to a miniDAWN MALS detector and an Optilab differential refractive index detector (dRI) (Wyatt Technology). For all experiments, a protein concentration of 2 mg mL−1, a flowrate of 0.8 mL min−1, an injection volume of 100 μL, and buffer C with the addition of 0.02% NaN3 were used. Data collection and analysis were performed with the ASTRA 8.0.2.5 software (Wyatt Technology). For the analysis of each run, the signal of the dRI detector was used for protein concentration determination. A bovine serum albumin (BSA) standard at 2 mg mL−1 was used for MALS detector normalization, correction of peak alignment, peak broadening, and reproducibility.
6
Molecular Weight Determination of hMLC1
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The molecular weight of the hMLC1 oligomer was determined using a Superdex-200 Increase 10/300 GL column coupled with a miniDAWN light scattering detector and an Optilab refractive index detector (Wyatt Technology). Purified hMLC1 proteins at a concentration of 1 mg ml−1 in 200 µl were loaded onto the column equilibrated with 150 mM NaCl, 20 mM HEPES, pH 7.5 and 2 mM DDM. Bovine serum albumin, CLC-ec1 and CLC-ec2 were used as the molecular weight standards. The data were analysed using ASTRA 8 software (Wyatt Technology).
7
Static Light Scattering Analysis of AcrIE4-F7
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Static light scattering data were obtained using a Superdex 75 Increase 10/300 GL column (GE Healthcare) coupled with a miniDAWN (3-angle) light scattering detector (Wyatt Technology) and an Optilab T-rEX refractive index detector (Wyatt Technology). The column was equilibrated with 20 mM HEPES, pH 7.0 and 150 mM NaCl. Then, 100 μl of AcrIE4-F7 (150 μM) was loaded onto the column at a flow rate of 0.5 ml/min at 25°C. The results were analyzed using the ASTRA 8 software (Wyatt Technology).
8
Characterization of SOA Protein Fragments
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SEC–MALS measurements were performed at 25 °C in 25 mM HEPES (pH 7.5), 500 mM NaCl and 1 mM DTT as the column buffer using a GE Healthcare Superdex 200 10/300 Increase column on an Agilent 1260 HPLC at a flow rate of 0.5 ml min–1. Loading concentrations were 200 µM for the SOA(1–112) and SOA(1–229) fragments and 11 µM for the SOA(1–331) fragment. Elution was monitored using an Agilent multi-wavelength absorbance detector (data collected at 280 and 260 nm), a Wyatt Heleos II 8+ multi-angle light scattering detector and a Wyatt Optilab differential refractive index detector. The column was equilibrated overnight in the running buffer to obtain stable baseline signals from the detectors before data collection. Inter-detector delay volumes, band-broadening corrections and light-scattering detector normalization were calibrated using an injection of 2 mg ml–1 BSA solution (Thermo Pierce) and standard protocols in ASTRA 8. Weight-averaged molar mass (Mw), elution concentration and mass distributions of the samples were calculated using ASTRA 8 software (Wyatt Technology).
9
Monodispersity Analysis of Tat86 Protein
10
SEC-MALS Analysis of BxpB Protein
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A 100-µL sample of BxpB was injected onto a WTC-MP015N5 column, 4.6 by 300 mm I.D; particle size, 5 mm; pore size, 150 Å (Wyatt Technology, Santa Barbara, CA) on a Shimadzu Prominence HPLC System (Shimadzu Corp., Kyoto, Japan) with an isocratic run at 0.3 mL/min for 17 min, and a solution of 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2 mM CaCl2, and 2 mM TCEP was used as the mobile phase. UV absorbance was set at 280 nm using Shimadzu HPLC Explorer software (Shimadzu Corp., Kyoto, Japan). A DAWN 8 MALS detector (Wyatt Technology, Santa Barbara, CA), set at 659 nm, and an Optilab refractometer (Wyatt Technology, Santa Barbara, CA) were used in tandem for detection. Bovine serum albumin (Wyatt Technology, Santa Barbara, CA) was used to normalize the static light scattering detector. The delay volume, band broadening parameters, and the light scattering and differential refractive index measurements were analyzed using Astra 8 software (Wyatt Technology, Santa Barbara, CA).
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