aF4 was hyphenated with a multi-angle laser light spectrometry (MALS) detector for determination of diameters of gyration of the fractionated TiMs dissolved at 250 mg L−1 to achieve appropriate signal intensity. The MALS detector (DAWN® HELEOS™, Wyatt Technology Europe GmbH, Dernbach, Germany) was operated with 17 + 1 observation angles and a linearly polarized laser beam at λ = 658 nm. Data acquisition was set at 2 s. The collected light scattering response was processed using ASTRA software (Wyatt, Dernbach, Germany) to calculate diameters of gyration (Øg), also known as root-mean-square diameter (= 2 × root-mean-square radius, rrms). For the nine TiMs investigated, fitting models, provided with the ASTRA software, were evaluated on three criteria: (1) pre-existing information on particle shape; (2) the calculated ratio of Øg/Øh, which gives indications on the particle shape. (3) It is expected that particle size increases linearly with retention time during aF4 fractionation. If this is indicated by the calculated Øg data, the selected model is able to appropriately reflect the particle elution behaviour. In the present study, all models were tested and the Berry model was selected according to the listed criteria.
Astra software
Manufactured by Wyatt Technology
Sourced in United States, Germany
ASTRA software is a powerful data analysis tool developed by Wyatt Technology. It is designed to provide accurate and reliable data processing and analysis for a variety of applications, including characterization of macromolecules and nanoparticles.
Automatically generated - may contain errors
Lab products found in correlation
Superdex 200 10 300 gl column, by GE Healthcare (35 mentions)
Minidawn treos, by Wyatt Technology (34 mentions)
Optilab t rex, by Wyatt Technology (33 mentions)
Dawn heleos 2, by Wyatt Technology (22 mentions)
Optilab rex, by Wyatt Technology (19 mentions)
Minidawn treos detector, by Wyatt Technology (16 mentions)
Optilab t rex refractive index detector, by Wyatt Technology (13 mentions)
Optilab t rex detector, by Wyatt Technology (12 mentions)
Dawn heleos, by Wyatt Technology (10 mentions)
Optilab rex refractive index detector, by Wyatt Technology (10 mentions)
Superose 6 increase 10 300 gl column, by GE Healthcare (9 mentions)
Superdex 200 increase 10 300 gl column, by GE Healthcare (9 mentions)
Dawn heleos 2 detector, by Wyatt Technology (8 mentions)
Optilab t rex refractometer, by Wyatt Technology (7 mentions)
Superdex 200 10 300 column, by GE Healthcare (7 mentions)
Minidawn treos light scattering detector, by Wyatt Technology (6 mentions)
Superdex 200 10 300 gl, by GE Healthcare (6 mentions)
Optilab t rex system, by Wyatt Technology (6 mentions)
Optilab t rex differential refractometer, by Wyatt Technology (6 mentions)
Akta purifier, by GE Healthcare (5 mentions)
357 protocols using astra software
1
TiMs Characterization by aF4-MALS
2
Determination of GCAP5 Molar Mass
3
SEC-MALS Analysis of Protein Samples
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Proteins of interest were diluted in SEC-MALS Buffer (50 mM HEPES pH 8.0, 1 mM EDTA and 200 mM NaCl) to a final concentration of 50 μM and centrifuged in a refrigerated microcentrifuge at 21 000 × g for 10 min to remove any aggregated protein. The supernatant was loaded onto a Superdex 200 Increase 10/300 GL (GE Healthcare) on an AKTA Pure FPLC system (GE Healthcare) with MALS being conducted using a MiniDAWN and Optilab system (Wyatt Technology). Data was collected and analysed using the Astra software, version 7.3.1.9 (Wyatt Technology) and plotted in Prism v.9.0 (GraphPad).
4
SEC-MALS Analysis of BTB Domains
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The SEC-MALS measurements were performed with MIZ1BTB, KAISOBTB, LRFBTB domains at 100 μM passed through the Superdex 200 30/300 column (GE Healthcare) using Agilent HPLC following DAWN® MALS detector. The running buffer contained 50 mM HEPES (pH 7.5), 150 mM NaCl, and 1 mM TCEP. Data were analyzed with the ASTRA® software provided by the company (Wyatt Technologies). The presented results are mean values with standard error from the three sample replicates.
5
Size Exclusion Chromatography of POEG3A
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Size exclusion chromatography (SEC) experiments were performed on an Agilent 1200 series HPLC system equipped with an Agilent PLgel mixed guard column (particle size = 5 µm) and two Agilent PLgel mixed-D columns (ID = 7.5 mm, L = 300 mm, particle size = 5 µm). Signals were recorded by a UV detector (Agilent 1200 series), an Optilab REX interferometric refractometer, and a miniDawn TREOS light scattering detector (Wyatt Technology Corp.). Samples were run using THF as the eluent at 30 °C and a flow rate of 1.0 mL/min. Data analyses were carried out on Astra software (Wyatt Technology Corp.) and molecular weights were determined based on narrow molecular weight poly(styrene) standards. Polymer 2 had a Mn = 6,700 g/mol , weight-average molecular weight (Mw) = 13,000 g/mol, dispersity (Ð) = 1.95. (see Supporting Information, Figure S6). The relatively high dispersity is common for atom transfer radical polymerization (ATRP) of OEG3A monomers without the introduction of CuX2 deactivator species. 47 (link) The deprotected POEG3A had a Mn = 5,100 g/mol, Mw = 10,000 g/mol, Ð = 1.95 (see Supporting Information, Figure S5 -S6, Table S1). However, in this article, the Mn of 17,700 g/mol determined by NMR is quoted, as this method gives a more accurate estimate of the molecular weight, than SEC with a polystyrene standard.
6
SEC-MALS Analysis of ANKS Proteins
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100 μL of protein (either ANKS3-SAM/ANKS6-SAM heterodimer at 15 mg/mL, ANKS6-SAM wt at 10 mg/mL, or ANKS6-SAM R823W at 10 mg/mL) was analysed by SEC-MALS. Protein was loaded onto a WTC-030S5 analytical size-exclusion column (Wyatt Technology Co.) equilibrated in 20 mM Tris pH 8.0, 0.15 M NaCl, (+2 mM BME for the ANKS3-SAM/ANKS6-SAM heterodimer) using an AKTA purifier (GE) and run at 0.5 mL/min on a miniDAWN TREOS (Wyatt Technology Co.). Eluted protein peaks were analysed for calculated molecular weight and monodispersity using ASTRA software (Wyatt Technology Co.)
7
SEC-MALS for Macromolecular Analysis
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Size exclusion chromatography–multiangle light scattering (SEC-MALS) measurements were performed using a DAWN HELEOS II MALS detector (Wyatt Technology, Santa Barbara, CA, USA) coupled to an Infinity II 1260 high-performance liquid chromatography system (Agilent Technologies, USA). For separation, a Superdex 200 Inc. 10/300 column (GE Healthcare, UK) was used with PBS as the mobile phase. Flow rates of either 0.5 or 0.75 ml min−1 were applied. Data were collected and analyzed in ASTRA software (Wyatt Technology, Santa Barbara, CA, USA).
8
Protein Characterization by SEC-MALS
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The purity, molecular mass, and size distribution of the proteins were analyzed by an analytical light scattering instrument (SEC-MALS) consisting of an Agilent 1260 Infinity Isocratic Liquid Chromatography System, a Wyatt Dawn Heleos II multiangle light scattering detector (Wyatt Technology), and a Wyatt Optilab T-rEX refractive index detector (Wyatt Technology). Analytical SEC was performed at about 25°C using a Superdex 200 10/300 GL column (GE Healthcare) equilibrated with a mobile phase containing 10 mM Hepes (pH 7.5), 200 mM NaCl, and 10 mM DTT. One hundred microliters of protein sample (10 mg/ml) was injected into the column and eluted at a flow rate of 0.5 ml/min. The column effluent was monitored in-line with three detectors that simultaneously monitor ultraviolet absorption, light scattering, and the refractive index. The measurements were analyzed using the ASTRA software (Wyatt Technology) to determine the molecular mass of the protein. The experiments were performed three times to check for reproducibility of the results.
9
SEC-MALS Analysis of NHLRC2 Domains
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Fragments of NHLRC2 containing the Trx-like and β-propeller domains (1–575) and the C-terminal domain (587–726) for SEC-MALS analysis were prepared by cleavage of the full-length protein with endoproteinase GluC (Roche) at 1:1000 (w/w) ratio overnight at room temperature. The formation of peptide fragments of the correct length was confirmed by mass spectrometry. Purified Trx-like domain with a linker region and β-propeller domain were mixed in equimolar ratio and incubated for 3 hours at room temperature. Full-length NHLRC2, the Trx-like domain, the β-propeller domain, an equimolar mixture of the Trx-like domain and the β-propeller domain, and full-length NHLRC2 after GluC treatment were sequentially loaded onto a Superdex 200 Increase 10/300 size exclusion column (GE Healthcare) pre-equilibrated with 20 mM BisTris pH 6.5, 150 mM NaCl, 2 mM DTT. The eluted samples first passed through a miniDAWN™ TREOS multi-angle light scattering detector (Wyatt technology) and then through a RID-10A refractometer (Shimadzu). The data were analyzed using ASTRA software (Wyatt Technology).
10
Molecular Mass Determination of ItcR IBD
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Molecular mass of purified ItcR itaconate-binding domain (IBD) was analyzed by an analytical light scattering instrument consisting of a 1260 infinity II LC system (Agilent Technology), a DAWN Heleos-II multiangle light scattering detector (Wyatt Technology), and an Optilab T-rEX refractive index detector (Wyatt Technology). Briefly, protein sample (100 μl) was loaded onto a HiLoad 10/300 Superdex 200 column (GE Healthcare) connecting to 1260 infinity II LC system and then eluted at a flow rate of 0.4 ml min−1. Column effluent was monitored simultaneously with three detectors for 280 nm ultraviolet light absorption, light scattering and refractive index. Data were analyzed by using ASTRA software (Wyatt Technology) to determine molecular mass of the protein.
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