Hundred microlitres of DIX samples were resolved on a Superdex-75 HR10/300 analytical gel filtration column (GE Healthcare) at 0.5 ml min−1 in 20 mM Tris (pH 7.4), 200 mM NaCl, 0.01% (w/v) NaN3 before light scattering (on a Wyatt Heleos II 18 angle light scattering instrument coupled to a Wyatt Optilab rEX online refractive index detector) in a standard SEC-MALS format. Heleos detector 12 was replaced with a Wyatt's QELS detector for dynamic light scattering measurements. Protein concentration was determined from the excess differential refractive index based on 0.186 RI increment for 1 g ml−1 protein solution. Concentrations and observed scattered intensities at each point in the chromatograms were used to calculate the radius of gyration and absolute molecular mass from the slope and the intercept of the Debye plot, using Zimm's model as implemented in Wyatt's ASTRA software. Autocorrelation analysis of data from the dynamic light scattering detector was also performed using Wyatt's ASTRA software, and the translational diffusion coefficients determined were used to calculate the hydrodynamic radius using the Stokes-Einstein equation.
Superdex 75 hr10 300
Manufactured by GE Healthcare
Sourced in France, United States
Superdex-75 HR10/300 is a size exclusion chromatography column designed for high-resolution separation of proteins and biomolecules with a molecular weight range of 3,000 to 70,000 Da. The column features a stable and rigid matrix that provides efficient separation and high resolution. It is suitable for analytical and preparative applications in life science research and development.
Automatically generated - may contain errors
Lab products found in correlation
Optilab rex, by Wyatt Technology (3 mentions)
Optilab rex refractometer, by Wyatt Technology (2 mentions)
659 nm laser beam, by Wyatt Technology (2 mentions)
Qels dynamic light scattering module, by Wyatt Technology (2 mentions)
Minidawn treos multi angle light scattering equipment, by Wyatt Technology (2 mentions)
Astra 7, by Wyatt Technology (2 mentions)
Heleos 2 18, by Wyatt Technology (1 mentions)
Heleos 2, by Wyatt Technology (1 mentions)
Nanostar, by Wyatt Technology (1 mentions)
Akta purifier 10, by GE Healthcare (1 mentions)
Pcr2.1 topo vector, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
8 protocols using superdex 75 hr10 300
1
SEC-MALS Analysis of Protein Complexes
2
Oligomer Formation Analysis of Arc Repressors
3
SEC-MALS Analysis of NTD Oligomerization
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NTD samples for SEC-MALS (100 µL at 5 mg/mL) were resolved on a Superdex-75 HR10/300 analytical gel filtration column (GE Healthcare) at 0.5 ml min−1 in either 50 mM phosphate, pH 7.0, with I adjusted to 200 mM using KCl (monomer conditions) or 20 mM MES, pH 6.0, with I adjusted to 60 mM using KCl (dimer conditions). Elution was detected using UV absorbance, light scattering with a Wyatt Heleos II 18 angle instrument, and finally refractive index using a Wyatt Optilab rEX instrument in a standard SEC-MALS format. Heleos detector 12 was replaced with a Wyatt’s QELS detector for dynamic light scattering measurements. Protein concentration was determined from the excess differential refractive index based on 0.186 RI increment for 1 mg/ml protein solution. Concentrations and observed scattered intensities at each point in the chromatograms were used to calculate the absolute molecular mass from the intercept of the Debye plot, using Zimm’s formalism as implemented in Wyatt’s ASTRA software.
4
Molecular Weight Determination of DCB Proteins
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MBP-tagged DCBBIG1 proteins were resolved on a Superdex-75 HR10/300 analytical gel filtration column (GE Healthcare) at 0.5 ml/min in PBS (pH 7.5), 150 mM NaCl, 5 mM MgCl2, and 0.001% (w/v) Triton X-100 before detection on a Wyatt Heleos II 18 angle light-scattering instrument coupled to a Wyatt Optilab rEX online refractive index detector and determination of native molecular weight (Perica et al., 2014 (link)).
5
Protein Purification and Characterization
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SEC experiments have been performed using a Superdex 75 HR10/300 analytic column (22 mL, exclusion limit of 70 kDa) with an AKTA Purifier10 chromatographic system (GE Healthcare, France). The detection was done by absorbance measurement at 215nm, 254nm and 280nm. Elution was performed at a flow rate of 0.450 mL/min with Tris-HCl 100 mM, NaCl 100 mM, glycerol 10%, pH 7.5 or TrisHCl 20 mM, NaCl 250 mM, glycerol 10%, pH 8 for proteins purified in denaturing or native conditions, respectively. Fractions of 250 μL were collected for analysis. Finally, Dynamic Light Scattering analysis has been performed using a NanoStar instrument (Wyatt Technology, France).
6
Cloning and Protein Expression Protocol
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Oligonucleotide primers were obtained from KRD (Prague, CZ, Czech Republic), Generi Biotech (Hradec Králové, Czech Republic, CZ) and Sigma-Aldrich (St. Louis, MO abbreviation, USA). Superscript III reverse transcriptase and the pCR2.1-TOPO vector were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The Escherichia coli BL-21 (DE3) Gold strain was from Stratagene (La Jolla, CA, USA), and the pET-30a(+) bacterial expression vectors were from Novagene (Madison, WI, USA). The pXJ41 vector was prepared by Xiao et al., 1991 [52 (link)]. Restriction endonucleases and other enzymes for DNA cloning were purchased from New England Biolabs (Ipswich, MA, USA) and Thermo Fisher Scientific (Waltham, MA, USA). The chromatographic column Superdex 75 HR 10/300 was obtained from GE Healthcare (Chicago, IL, USA). All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise declared and were of the highest available purity.
7
SEC-MALS Analysis of Protein Samples
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SEC-MALS measurements were performed on a miniDAWN TREOS multi-angle light scattering equipment with detectors at three angles (43.6°, 90° and 136.4°) and a 659 nm laser beam (Wyatt Technology, CA). A Wyatt QELS dynamic light scattering module for determination of hydrodynamic radius and an Optilab® rEX refractometer (Wyatt Technology)
were used in-line with a size exclusion chromatography analytical column (Superdex 75 HR10/300, GE Healthcare). BSA was used as a control. The protein solutions (~2 mg/mL) were eluted in a 50 mM Tris HCl, 500 mM NaCl buffer, pH 8.0 with a flow rate of 0.5 mL/min. The data were processed using ASTRA7 software (Wyatt Technology) with the following parameters:
refractive index of 1.331, 0.890 cP for the viscosity of the solvent and a refractive index increment of 0.1850 mL/g. Protein solutions were centrifuged for 10 minutes at 10,000xg at 4°C prior to use.
were used in-line with a size exclusion chromatography analytical column (Superdex 75 HR10/300, GE Healthcare). BSA was used as a control. The protein solutions (~2 mg/mL) were eluted in a 50 mM Tris HCl, 500 mM NaCl buffer, pH 8.0 with a flow rate of 0.5 mL/min. The data were processed using ASTRA7 software (Wyatt Technology) with the following parameters:
refractive index of 1.331, 0.890 cP for the viscosity of the solvent and a refractive index increment of 0.1850 mL/g. Protein solutions were centrifuged for 10 minutes at 10,000xg at 4°C prior to use.
8
SEC-MALS Analysis of Protein Samples
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SEC-MALS measurements were performed on a miniDAWN TREOS multi-angle light scattering equipment with detectors at three angles (43.6°, 90° and 136.4°) and a 659 nm laser beam (Wyatt Technology, CA). A Wyatt QELS dynamic light scattering module for determination of hydrodynamic radius and an Optilab® rEX refractometer (Wyatt Technology)
were used in-line with a size exclusion chromatography analytical column (Superdex 75 HR10/300, GE Healthcare). BSA was used as a control. The protein solutions (~2 mg/mL) were eluted in a 50 mM Tris HCl, 500 mM NaCl buffer, pH 8.0 with a flow rate of 0.5 mL/min. The data were processed using ASTRA7 software (Wyatt Technology) with the following parameters:
refractive index of 1.331, 0.890 cP for the viscosity of the solvent and a refractive index increment of 0.1850 mL/g. Protein solutions were centrifuged for 10 minutes at 10,000xg at 4°C prior to use.
were used in-line with a size exclusion chromatography analytical column (Superdex 75 HR10/300, GE Healthcare). BSA was used as a control. The protein solutions (~2 mg/mL) were eluted in a 50 mM Tris HCl, 500 mM NaCl buffer, pH 8.0 with a flow rate of 0.5 mL/min. The data were processed using ASTRA7 software (Wyatt Technology) with the following parameters:
refractive index of 1.331, 0.890 cP for the viscosity of the solvent and a refractive index increment of 0.1850 mL/g. Protein solutions were centrifuged for 10 minutes at 10,000xg at 4°C prior to use.
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