SEC–multiangle light scattering (MALS) measurements of LicB were performed at 18°C in 10 mM tris-HCl (pH 8.0), 150 NaCl, and 0.012% LMNG using a GE Healthcare Superdex 200 Increase 10/300 GL column on an Agilent 1260 high-performance liquid chromatography. The column was equilibrated overnight for a stable baseline before data collection. Monitoring of the elution was carried out with a multiwavelength absorbance detector at 280 and 254 nm, a Wyatt Heleos II 8+ MALS detector, and a Wyatt Optilab rEX differential refractive index detector. Interdetector delay volumes, light scattering detector normalization, and broadening corrections were calibrated using a bovine serum albumin solution (2 mg/ml; ThermoPierce) and standard protocols in ASTRA 6 (Wyatt Technologies). Weight-averaged molar mass, elution concentration, and mass distribution of the samples were calculated using the ASTRA 6 software (Wyatt Technologies). The specific refractive index increment for the detergent LMNG was assumed to be 0.146 mg/ml according to experimental data.
Astra 6
Manufactured by Wyatt Technology
Sourced in United States, Germany
The ASTRA 6 software is a powerful analytical tool used for the characterization of macromolecules and nanoparticles. It provides comprehensive data analysis capabilities for a variety of light scattering techniques, including static and dynamic light scattering, as well as multi-angle light scattering. The software offers advanced algorithms and intuitive user interface to enable efficient data processing and interpretation.
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Lab products found in correlation
Optilab t rex, by Wyatt Technology (88 mentions)
Dawn heleos 2, by Wyatt Technology (40 mentions)
Optilab t rex differential refractometer, by Wyatt Technology (34 mentions)
Dawn heleos 2 mals detector, by Wyatt Technology (29 mentions)
Minidawn treos, by Wyatt Technology (28 mentions)
Superdex 200 increase 10 300 gl column, by GE Healthcare (23 mentions)
Optilab t rex refractive index detector, by Wyatt Technology (23 mentions)
Superdex 200 10 300 gl column, by GE Healthcare (20 mentions)
Dawn 8, by Wyatt Technology (19 mentions)
Optilab rex, by Wyatt Technology (17 mentions)
Superdex 200 increase 10 300 gl, by GE Healthcare (16 mentions)
Kta pure, by GE Healthcare (15 mentions)
Superose 12 10 300 gl column, by GE Healthcare (13 mentions)
Superdex 200 increase 10 300 column, by GE Healthcare (13 mentions)
Optilab t rex refractometer, by Wyatt Technology (13 mentions)
Dawn heleos 2 detector, by Wyatt Technology (13 mentions)
Superdex 200 10 300 gl, by GE Healthcare (11 mentions)
Akta fplc system, by GE Healthcare (10 mentions)
Dawn heleos 2 eos 18 angle laser photometer, by Wyatt Technology (10 mentions)
Minidawn treos detector, by Wyatt Technology (9 mentions)
413 protocols using astra 6
1
SEC-MALS Analysis of LicB Protein
2
Molecular Weight Determination by SEC
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The size exclusion chromatography was carried out using a WATERS E2695 chromatograph (Waters, Waters Corporation, Milford, MA, USA) equipped with HR-1, HR-3 and HR-4 columns (500–500,000 g·mol−1) and coupled with a differential refractometer (Optilab®-T-rEX, Wyatt Technology, Santa Barbara, CA, USA). The system is calibrated using a standard polystyrene solution. 15 mg of sample was dissolved in 3 mL of THF (n = 6). 1mL of toluene per liter of THF was added. Subsequently, these test solutions were filtered through a PTFE membrane with a pore diameter of 0.45 μm and then transferred to glass vials. Data processing was carried out using Astra 6 software (Astra 6, Wyatt Technology, USA).
3
SEC-MALS Analysis of Recombinant ATG14
4
SEC-MALS Analysis of DmpR Oligomerization
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MALS analysis was performed using a WTC-050S5 SEC column with an in-line Dawn Helios II system and an Optilab T-rEX differential refractometer (Wyatt). DmpR (10 µM), phenol (1 mM) and/or ATP/ATPγS (3 mM) were incubated at 25 °C for 20 min in PBS buffer. After centrifugation, the supernatant was applied to a SEC-MALS system with PBS elution buffer containing 0.5 mM phenol. The data were collected and analysed using ASTRA 6 (Wyatt). Gradient gels (4–16%) were used for BN-PAGE (Novex). To identify factors that might influence the oligomer state of DmpR, 20 µM DmpR, was incubated for 20 min at 25 °C in the presence or absence of 1 mM phenol, 5 mM MgCl2 and/or 3 mM ATP, respectively. To determine change of DmpR tetramer by ATP analogue or UAS containing DNA, 3 mM ATP analogue (AMP-PNP/ATPγS), 10 nM DNaseI (NEB) or 20 µM cognate DNA with the UAS sites were co-incubated with DmpR for 20 minutes prior to BN-PAGE analysis.
5
SEC-MALS Analysis of Pf4r and Pf6r
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SEC-MALS was performed according to previously established methods [25 (link)]. Briefly, Pf4r, Pf4r* and Pf6r samples (prepared in 20 mM HEPES pH 7.5, 300 mM NaCl, glycerol 10% (v/v), 2mM TCEP) were gel-filtrated on a Superdex 75 5/150 gel filtration column (GE Healthcare, IL, USA) equilibrated with PBS. The chromatography system was connected in-line to a miniDAWN light scattering unit (Wyatt Technology, CA, USA) and an Optilab T-rEX differential refractive index detector (Wyatt Technology, CA, USA). Loading volumes were 50 μL with protein concentrations of 20 mg/mL. Data were analysed in Astra 6 (Wyatt Technology, CA, USA).
6
SEC-MALS Analysis of Protein Complexes
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Proteins were mixed at the indicated ratios and equilibrated in 50 mM HEPES pH 7.5, 100 mM NaCl, 1 mM MgCl2, 1 mM Dithiothreitol (DTT) and 10% glycerol (v/v) buffer supplemented with 1 mM adenosine diphosphate (ADP), ATP or AMPPNP for 3 h at room temperature. A total of 100 μl of these mixtures (containing 50–150 μg of total protein) were loaded onto either a Superose 6 HR10/30 column, or a Superdex 200 HR10/30 column (GE), equilibrated with the same buffer lacking glycerol and nucleotides. The separation was conducted at a flow rate of 0.5 ml/min. Presence of DTT or Tris(2-carboxyethyl)phosphine (TCEP) as reductants did not influence the results. Size exclusion chromatography and multi-angle light scattering (SEC-MALS) analysis was performed at 22°C using a Shimadzu (Kyoto, Japan) chromatography system, connected in-line to a Heleos8+ multi angle light scattering detector and an Optilab T-rEX refractive index (RI) detector (Wyatt Technologies, Goleta, CA, USA). Results were processed and analysed using ASTRA 6 (Wyatt Technologies).
7
SEC-MALLS Analysis of Timeless Proteins
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For SEC-MALLS experiments a Superdex 200 Increase 10/300 column (GE Healthcare) was coupled with a DAWN HELEOS II MALLS detector with a 664 nm laser light source, eight fixed-angle detectors and an Optilab T-rEX differential refractometer (Wyatt Technology) at 25°C. A total of 1–2 mg/ml (100 μl injection) of Timeless(1–1208Δ239-330) and Timeless(1–463Δ239-330) were analysed in 25 mM Hepes 7.2, 150 mM KCl, 50 mM Arginine, 50 mM Glutamate. The collected data were analysed and processed using ASTRA 6 (Wyatt Technology).
8
Determining Protein MW by SEC-MALS
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Proteins in 50 mM HEPES pH 7.5 with 100 mM NaCl were studied by analytical size-exclusion chromatography on a WTC-050S5 column (Wyatt Technology) and directly flowed into a Wyatt DAWN HELEOS II light-scattering detector and a Wyatt Optilab T-rEX refractive-index detector (Wyatt Technology). The column was employed to determine the average molecular mass of the elution peak from the Rayleigh scattering intensity as a function of the scattering index (LSR) and the buffer scattering index (dRI) using ASTRA 6 (Wyatt Technologies) (Trathnigg, 1995 ▸ ).
9
SEC-MALS Analysis of Purified Complexes
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The SEC-MALS system used was an AKTApure FPLC (GE), with an in-line Optilab T-Rex refractometer (Wyatt), and a Dawn Heleos II light scattering instrument (Wyatt). 500 μL of purified complex (~2 mg/mL) was injected onto a WTC-050S5 SEC column (Wyatt) and eluted at 0.5 mL/min directly into the on-line MALS instruments. Data was collected and processed to determine molecular mass using Astra 6 (Wyatt).
10
Protein Characterization by SEC-MALS
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The analysis was performed on an AKTA FPLC system (GE Healthcare) coupled with a static light scattering detector (miniDawn, Wyatt) and a differential refractive index detector (Optilab, Wyatt). Protein samples (70 μM for E-cadherin and 70 μM or 140 μM or 210 μM or 280 μM for AnkG) were filtered and loaded into a Superdex 200 increase column pre-equilibrated by a column buffer composed of 50 mM Tris, 100 mM NaCl, 1 mM EDTA, and 1 mM DTT at pH 7.8. Data were analyzed with ASTRA6 (Wyatt).
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