SEC-MALLS experiments for molecular weight determination of DdrO and its domains was carried out with an analytical Superdex-200 increase column (GE Healthcare) equilibrated with 50 mM Tris–HCl pH 7.4, 150 mM NaCl. Runs were performed at 25°C with a flow rate of 0.5 ml/min. Elutions were monitored by using a DAWN-EOS detector with a laser emitting at 690 nm for online MALLS measurement (Wyatt Technology Corp., Santa Barbara, CA, USA) and with a RI2000 detector for online refractive index measurements (Schambeck SFD). Molecular weight calculations were performed using the ASTRA software (version 5.3.4.20) using a refractive index increment (dn/dc) of 0.185 ml/g.
Dawn eos detector
Manufactured by Wyatt Technology
Sourced in United States
The DAWN-EOS detector is a laser light scattering instrument designed for the analysis of macromolecules and nanoparticles. It measures the intensity of scattered light at multiple angles to determine the molar mass, size, and shape of samples in solution.
Automatically generated - may contain errors
Lab products found in correlation
Astra 5, by Wyatt Technology (3 mentions)
Astra software, by Wyatt Technology (2 mentions)
Superdex 200 increase column, by GE Healthcare (1 mentions)
Superdex 200 10 300 gl column, by GE Healthcare (1 mentions)
Gel filtration calibration kit, by GE Healthcare (1 mentions)
Superdex 75 10 300 gl, by GE Healthcare (1 mentions)
Optilab rex refractive index detector, by Wyatt Technology (1 mentions)
Superdex 200 increase 10 300 gl column, by Cytiva (1 mentions)
1200 pump, by Agilent Technologies (1 mentions)
J 810 spectropolarimeter, by Jasco (1 mentions)
Nanosep centrifugal device, by Pall Corporation (1 mentions)
11 protocols using dawn eos detector
1
SEC-MALLS for Determining Molecular Weight
2
Molecular Mass Analysis of LFY Proteins
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The molecular mass of GbLFY-SAM and AtLFY-SAM was estimated at 4 °C using a Superdex-200 10/300GL column (GE Healthcare), equilibrated with buffer A and calibrated with low- and high-molecular-weight protein standards (gel filtration calibration kit; GE Healthcare). Accurate molecular mass determination using SEC-MALLS was carried out with a Superdex-200 10/300GL column (GE Healthcare). GbLFY-SAM, WT and mutants were analysed in buffer A. Protein–DNA complexes containing GbLFYΔ, WT or mutants and AP1 DNA were analysed in 20 mM Tris-HCl pH 8, 150 mM NaCl, 0.25 mM EDTA, 2 mM MgCl2 and 1 mM TCEP. Separations were performed at 20 °C with a flow rate of 0.5 ml min−1. Elutions were monitored by using a DAWN-EOS detector with a laser emitting at 690 nm for online MALLS measurement (Wyatt Technology Corp., Santa Barbara, CA) and with a RI2000 detector for online refractive index measurements (Schambeck SFD). Molecular mass calculations were performed using the ASTRA software using a refractive index increment (dn/dc) of 0.185 ml g−1.
3
Size-Exclusion Chromatography for Protein Analysis
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Size-Exclusion Chromatography experiments (SEC) was performed with a Superdex 200 analytic Increase (GE Healthcare) equilibrated in 100 mM Tris-Cl pH 8, 200 mM NaCl, 5 mM TCEP. Calibration has been performed using bovine serum albumin (BSA). Separations were performed at 20°C with a flow rate of 0.5 ml.min−1. Note that 50 μl of a protein solution at a concentration of 7 mg.ml−1 were injected. Online MALLS detection was performed with a DAWN-EOS detector (Wyatt Technology Corp., Santa Barbara, CA,USA) using a laser emitting at 690 nm. Protein concentration was measured online by refractive index measurements using a RI2000 detector (Schambeck SFD) and a refractive index increment 0.185 ml.g−1. Data were analyzed and weight-averaged molecular masses (Mw) were calculated using the software ASTRA V (Wyatt Technology Corp., Santa Barbara, CA, USA).
4
Protein Molecular Mass Determination by SEC-MALLS
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SEC was performed with a Superdex 75 10/300 GL (GE Healthcare) equilibrated in 50 mM Tris-HCl pH 7.5, 100 mM NaCl. Separations were performed at 20°C with a flow rate of 0.5 mL.min−1. 50 µL of a protein solution at a concentration of 10 mg.mL−1 were injected. On-line MALLS detection was performed with a DAWN-EOS detector (Wyatt Technology Corp., Santa Barbara, CA) using a laser emitting at 690 nm. Protein concentration was measured on-line by refractive index measurements using a RI2000 detector (Schambeck SFD) and a refractive index increment dn/dc = 0.185 mL.g−1. Data were analyzed and weight-averaged molecular masses (Mw) were calculated using the software ASTRA V (Wyatt Technology Corp., Santa Barbara, CA) as described previously [40] (link).
5
SEC-MALLS Analysis of MsVps4 Phe126Ala
6
SEC-MALS Characterization of Proteins
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SEC was performed on a HPLC system (1200 pump, Agilent Technologies) using a precalibrated Superdex 200 increase 10/300 GL column (Cytiva) run in 20 mM Tris-HCl pH8, 1 M NaCl, 10% glycerol, and 5 mM DTT at 0.5 ml/min. MALS, UV spectrophotometry, QELS, and RI were achieved with a DAWN EOS detector (Wyatt Technology), a SpectraSYSTEM UV2000 UV/VIS detector (Thermo Finnigan), a WYATT QELS module (Wyatt Technology), and an Optilab rEX Refractive Index detector (Wyatt Technology), respectively. Mass and hydrodynamic radius calculation was done with ASTRA 5.3 software (Wyatt Technology) using a dn/dc value of 0.175 ml/g, calculated as described (64 (link)).
7
SEC Analysis of MsVps4 Phe126Ala
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SEC was performed with a Superose 6. The column was equilibrated in 50 mM Tris pH 8.8 and 50 mM NaCl, and MsVps4 Phe126Ala was injected at 200 μM. The experiment was performed with MALLS using a DAWN-EOS detector (Wyatt Technology Corp., Santa Barbara, CA) and refractive index measurement using a RI2000 detector (Schambeck SFD). Light-scattering intensities were measured at different angles relative to the incident beam and analysis of the data was performed with the ASTRA software (Wyatt Technology Corp.).
8
SEC-MALLS Analysis of Acetylated p300
9
SEC-MALLS Analysis of Acetylated p300
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Prior to SEC-MALLS runs, p300 variants were acetylated and deacetylated using p300 HAT or SIRT2 as described previously26 (link). The reactions were analyzed by liquid chromatography-mass spectrometry (LC-MS) as done previously52 (link). Size-exclusion chromatography was performed at a flow rate of 0.5 ml min-1 on a Superdex 200 Increase 10/300 GL column equilibrated in SEC-MALLS buffer (20 mM HEPES, 300mM NaCl, 5μM ZnCl2, 0.5mM TCEP) at 21 °C. A 50 μl sample of p300 at 2 mg ml-1 was injected onto the column and multi angle laser light scattering was recorded with a laser emitting at 690 nm using a DAWN-EOS detector (Wyatt TechnologyCorp. Santa Barbara, CA). The refractive index was measured using a RI2000 detector (Schambeck SFD). The molecular weight was calculated form differential refractive index measurements across the center of the elution peaks using the Debye model for protein using ASTRA software version 6.0.5.3.
10
SEC-MALLS Analysis of TAZ2, NUT4, and Complex
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SEC-MALLS was performed at a flow rate of 0.5 ml/min on a Superdex 75 Increase 10/300 GL column equilibrated in buffer (20 mM HEPES pH7.0, 300 mM NaCl, 5 µM ZnSO4, 0.5 mM TCEP). 50 µl of TAZ2, NUT4 or the TAZ2:NUT4 complex at 2 mg/ml were injected onto the column and multi angle laser light scattering was recorded with a laser emitting at 690 nm using a DAWN-EOS detector (Wyatt Technology Corp). The refractive index was measured using a RI2000 detector (Schambeck SFD). The molecular weight was calculated from differential refractive index measurements across the centre of the elution peaks using the Debye model for protein using ASTRA software version 6.0.5.3.
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