SEC-MALLS experiments were run in 20 mM HEPES 7.4, 300 mM NaCl buffer. The injected sample comprised 100 µL of EnvSia156 at 1.8 mg mL−1 in 20 mM HEPES pH 7.4, 100 mM NaCl, 1 mM DTT. Experiments were conducted on a system comprising a Superdex 200 10/30 GL (GE Healthcare) size exclusion chromatography column, a Wyatt HELEOS-II multi-angle light scattering detector and a Wyatt rEX refractive index detector linked to a Shimadzu HPLC system (SPD-20A UV detector, LC20-AD isocratic pump system, DGU-20A3 degasser, and SIL-20A autosampler). Work was conducted at room temperature (20 ± 2 °C). All solvents and buffers were 0.2 µm filtered before use and a further 0.1 µm filter was present in the flow path. Shimadzu LC Solutions software was used to control the HPLC and Astra V software for the HELEOS-II and rEX detectors. All data were analyzed using the Astra V software. Molecular masses were estimated using the Zimm fit method with degree 1. A value of 0.16 mL g−1 was used for protein refractive index increment (dn/dc), after calibration with a 2.5 mg mL−1 sample of BSA.
Superdex 200 10 30 gl
Manufactured by GE Healthcare
Superdex 200 10/30 GL is a size exclusion chromatography column used for the purification and analysis of proteins, peptides, and other biomolecules. It is packed with a porous agarose-based medium that separates molecules based on their size and shape. The column has a bed volume of 24 mL and is designed for use with ÄKTA chromatography systems.
Automatically generated - may contain errors
Lab products found in correlation
Heleos 2, by Wyatt Technology (1 mentions)
Lc20 ad isocratic pump system, by Shimadzu (1 mentions)
Sil 20a autosampler, by Shimadzu (1 mentions)
Dgu 20a3 degasser, by Shimadzu (1 mentions)
Spd 20a uv detector, by Shimadzu (1 mentions)
Gel filtration standard, by Bio-Rad (1 mentions)
Mnase, by Worthington (1 mentions)
Amicon ultra 4 10 kda, by Merck Group (1 mentions)
Myoglobin, by Bio-Rad (1 mentions)
γ globulin, by Bio-Rad (1 mentions)
Vitamin b12, by Bio-Rad (1 mentions)
Thyroglobulin, by Bio-Rad (1 mentions)
Ovalbumin (ova), by Bio-Rad (1 mentions)
7 protocols using superdex 200 10 30 gl
1
SEC-MALLS Analysis of EnvSia156
2
Purification and Characterization of RhVI1
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The solubilized membranes, already enriched in RhVI1, were diluted 50 times in order to bring the final DDM concentration to 0.4mM (0.02% m/v). This diluted pool was poured into an AMICON 9000 stirred cell, coupled to a 100kDa cut-off ultrafiltration membrane, and concentrated to a final volume of 10ml. Finally, the volume was further concentrated to 500 µl using a Vivaspin 20 ultrafiltration with the same cut-off.
The protein sample was loaded on to a gel filtration column (Superdex 200 10/30 GL, GE Healthcare) pre-equilibrated with gel filtration buffer [20mM MES–NaOH, pH 6.5; 5mM CaCl2; 5mM MgCl2; 10mM NaHCO3; 0.01% (w/v) β-DDM]. The sample, the main peak and the related fractions, was pooled and concentrated by ultrafiltration (Vivaspin 20, 100kDa cut-off). The molecular weight of the RhVI1 was estimated by plotting the elution volume versus the logarithm of the molecular weight of the standard proteins (Gel Filtration Standard, Biorad) using a linear regression curve fit.
The protein sample was loaded on to a gel filtration column (Superdex 200 10/30 GL, GE Healthcare) pre-equilibrated with gel filtration buffer [20mM MES–NaOH, pH 6.5; 5mM CaCl2; 5mM MgCl2; 10mM NaHCO3; 0.01% (w/v) β-DDM]. The sample, the main peak and the related fractions, was pooled and concentrated by ultrafiltration (Vivaspin 20, 100kDa cut-off). The molecular weight of the RhVI1 was estimated by plotting the elution volume versus the logarithm of the molecular weight of the standard proteins (Gel Filtration Standard, Biorad) using a linear regression curve fit.
3
Histone Octamer Formation and Nucleosome Reconstitution
4
Purification of recombinant human GBP1
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Full-length human GBP1 was cloned in pET-28a to generate an N-terminally His-tagged hGBP1 construct. pET-28a-hGBP1 was transformed into CleanColi BL21 (Lucigen), and the bacteria were grown in 2xYT medium until an OD600 of 0.5-0.7. Protein expression was then induced at 30 °C for 5 h with 0.2 mM IPTG. The bacterial pellet was resuspended in resuspension buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Tween 20) and frozen at −80 °C until purification. For most assay, protein was freshly purified on a Ni-NTA affinity column using standard protocols48 (link). Protein yield was quantified using Beer-Lambert law. After purification on a Ni-NTA columns, GBP1 was further purified on a size exclusion chromatography column (Superdex 200 10/30 GL, GE Healthcare) in running buffer (50 mM Tris pH 7.4, 150 mM NaCl) and concentrated using Amicon Ultra4 10 kDa (Millipore).
5
Molecular Weight Determination of AxeA and AxeB
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Molecular weights of native AxeA and AxeB proteins were determined with Superdex™ 200 10/30 GL (GE, Healthcare) pre-equilibrated with 50 mM Tris–HCl buffer (pH 7.5) containing 150 mM NaCl. The following molecules were used as reference standards: thyroglobulin (670,000 Da), γ-globulin (160,000 Da), oval bumin (44,000 Da), myoglobin (17,000 Da) and vitamin B12 (135 Da) (Bio-Rad, USA).
6
Oligomeric State Determination of mGold
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Size exclusion chromatography was conducted to determine the oligomeric state of mGold. mGold (10 μM) and size standards—mCherry (monomeric, 10 μM), mVenus (monomeric, 10 μM), and tdTomato (dimeric, 6.5 μM)—were analyzed using a fast protein liquid chromatography instrument (NGC Chromatography, Bio-Rad) with a 30 cm × 10 cm (length × diameter) gel filtration column (Superdex 200 10/30 GL, GE Healthcare). One hundred microliters of samples were injected and ran separately at a flow rate of 0.5 ml/min. The flow buffer was 50 mM tris buffer at pH 7.5 supplemented with 100 mM NaCl. The FPs were detected by measuring the absorbance at 515 nm (mGold and mVenus), 587 nm (mCherry), and 555 nm (tdTomato).
7
Fluorescent Labeling of Proteins
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Labeling was done by adding 20-fold molar excess of MTSSL (100 mM, in DMF) to the eluted protein. The sample was then incubated at room temperature (RT) for 1.5 h after which 20-fold molar excess of MTSSL was added again, and the protein was incubated for an additional 1 h at RT. The sample was kept on ice overnight. The protein was loaded onto Superdex 200 10/30 GL (GE Healthcare) size exclusion chromatography column in 20 mM tris-HCl pH 7.5, 120 mM NaCl, 10% glycerol, 0.03% DDM. The protein was then concentrated to 75–100 μM using a 100 K MWCO concentrator (Amicon) and glycerol was added to a final concentration of 23.78% (v/v). SEC elution profiles are shown in Fig. S12 . In each case, the major monodispersed fraction was collected and used for the experiment.
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