All experiments were performed on a Superdex S75 10/300 GL column (GE Healthcare) with a sample injection volume of 250 μL and a flow rate of 0.5 mL/min at 4°C. The volume of the sample loop was 100 μL. Molecular weight markers used to calibrate the column were: albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), and ribonuclease A (13.7 kDa) from the Low Molecular Weight Gel-filtration Calibration kit (GE Healthcare). Absolute molecular weight calculations were obtained by static light scattering in line with size exclusion chromatography using the Wyatt Optilab T-rEX refractometer and miniDAWN Treos multiangle light scattering system at 4°C. Protein concentrations were monitored by the refractometer and light scattering unit directly after the gel filtration column. Absolute molecular weights were determined using ASTRA version 6.0 (Wyatt Technologies).
Superdex s75 10 300 gl column
Manufactured by GE Healthcare
The Superdex S75 10/300 GL column is a size exclusion chromatography column designed for the separation and purification of proteins, peptides, and other biomolecules. It has a separation range of 3,000 to 70,000 daltons and a bed volume of 24 ml. The column is made of high-quality materials and is suitable for use in a variety of applications, including protein analysis, protein purification, and sample preparation.
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Lab products found in correlation
Ni nitrilotriacetic acid nta column, by Qiagen (2 mentions)
Microfluidize m 110p, by Microfluidics (2 mentions)
Astra version 6, by Wyatt Technology (1 mentions)
Optilab t rex refractometer, by Wyatt Technology (1 mentions)
Low molecular weight gel filtration calibration kit, by GE Healthcare (1 mentions)
Waters 626 lc system, by Waters Corporation (1 mentions)
626 lc system, by Waters Corporation (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
7 protocols using superdex s75 10 300 gl column
1
Protein Molecular Weight Analysis
2
Analytical Gel Filtration of Recombinant Proteins
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Analytical gel filtration experiments of recombinant SIVmac239 Nef, HckSH3-E, and CD3 ζ fragments were performed with a multicomponent Waters 626 LC system (Waters, MA) connected to a Superdex S75 (10/300 GL) column (GE Healthcare). Typically, 100 µl of a 1.5 mg ml−1 protein solution was injected onto the column that was previously equilibrated with 10 mM Tris/HCl (pH 9.0), 100 mM NaCl buffer. Analytical gel filtration runs were done at a flow rate of 0.5 ml per minute at 293 K. The optical density was monitored at a wavelength of 280 nm. Gel filtration experiments were performed repeated times.
3
Analytical SEC Analysis of REV7-RINN1 Complex
4
Purification of His-tagged LytA Proteins
5
Analytical Gel Filtration of Recombinant Proteins
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Analytical gel filtrations of recombinant NefSF2, SH3B6, sdAb19, Neffin, and all mutants thereof were performed using a multicomponent Waters 626 LC system (Waters, MA) equipped with a Superdex S75 (10/300 GL) column (GE Healthcare). Typically, 100 μl of a 1.5 mg/ml protein solution was loaded onto the column that was equilibrated in 10 mM Tris/HCl (pH 9.0), 100 mM NaCl buffer prior to injection of the protein samples. Gel filtrations were run at a flow rate of 0.5 ml per minute in 10 mM Tris/HCl (pH 9.0), 100 mM NaCl onto the S75 column at 4°C or 20°C. The optical density was monitored at a wavelength of 280 nm over the time course of the experiment. Gel filtration experiments were performed repeated times.
6
Purification of His-tagged LytA Protein
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Strain BL21(DE3) Rosetta pLysS Rare (CmR) of E. coli was transformed with the plasmids pET28-His-LytA (KanR), pADG14 (KanR), pADG141 (KanR), or pADG142 (KanR). Cells were grown in Luria Bertani medium and protein expression was induced at OD595nm 0.6 with 0.5 mM IPTG (isopropyl β-D-thiogalactopyranoside) at 25 °C overnight. Cells from 2-litres cultures were harvested by centrifugation, resuspended in 50 ml of a buffer containing 50 mM Tris pH 8.0, 500 mM NaCl, 25 mM imidazole, 10% glycerol and a protease inhibitor cocktail (Complete EDTA free, Sigma–Aldrich) and lyzed using a Microfluidize M-110P (Microfluidics). The lysate was clarified by centrifugation (20 min at 39,191 × g at 4 °C) and loaded onto a 10-ml Ni-nitrilotriacetic acid (NTA) column (Qiagen). His-LytA proteins were eluted with a 25 mM to 500 mM imidazole gradient. Pooled fractions were concentrated and further purified by size exclusion chromatography using a Superdex S75 10/300 GL column (GE Healthcare) in 25 mM Tris-HCl (pH 8) and 150 mM NaCl.
7
Recombinant ICab Protein Production
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Generation and purification of ICab were carried out in a manner described previously24 (link). The ICab isolated through yeast-display screening was subcloned into the pET-22b vector. E. coli Rosetta cells were transformed with a vhh-containing vector. Large scale cultures were grown in LB broth at 37 °C until OD600 reached 0.6 and protein expression was induced by adding IPTG to a final concentration of 0.5 mM; cells were further grown at 37 °C for 5–6 h. The cells were harvested and the protein was extracted from inclusion bodies using urea denaturation followed by refolding. The refolding was done using step-wise dialysis at 4 °C. The protein was further purified by size-exclusion chromatography using Superdex S-75 10/300 GL column (GE Healthcare) in HBS. The purity and integrity of the protein were checked by SDS-PAGE and MALDI-TOF.
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