SEC-MALS experiments were performed by using SEC on an analytical Superdex S200 10/300 GL column (GE Healthcare Life Sciences) connected to online static light-scattering (DAWN HELEOS II, Wyatt Technology, Santa Barbara, CA), differential refractive index (Optilab rEX, Wyatt Technology, Santa Barbara, CA) and Agilent 1200 UV (Agilent Technologies, Santa Clara, CA) detectors. Purified sample (FzCRD proteins at 50 μM or Norrin–Fz4CRD complex at 25 μM) was injected into a column equilibrated in 10 mM HEPES, pH 7.5, 0.15 mM NaCl. Molecular mass determination was performed using an adapted RI increment value (dn/dc standard value; 0.186 ml/g) to account for the glycosylation state. The theoretical molecular weight was predicated from amino acid sequence plus 2.35 kDa per N-linked glycosylation site for full glycosylated protein produced from HEK293T cells or 203 Da per site for deglycosylated protein produced from HEK293T cells in the presence of kifunensine (Chang et al., 2007 (link)) with limited glycosylation and treated with Endo-F1. Data were analyzed using the ASTRA software package (Wyatt Technology, Santa Barbara, CA).
Superdex s200 10 300 gl column
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Switzerland
The Superdex S200 10/300 GL column is a size exclusion chromatography column used for the separation and purification of proteins and other macromolecules. It has a bed volume of 24 mL and is compatible with aqueous buffers and organic solvents.
Automatically generated - may contain errors
Lab products found in correlation
Optilab rex, by Wyatt Technology (2 mentions)
Astra 6, by Wyatt Technology (2 mentions)
Minidawn treos detector, by Wyatt Technology (2 mentions)
Dawn heleos 2, by Wyatt Technology (1 mentions)
Astra software package, by Wyatt Technology (1 mentions)
Agilent 1200 uv, by Agilent Technologies (1 mentions)
Seed bead, by Hampton Research (1 mentions)
Nativepage novex bis tris gel system, by Thermo Fisher Scientific (1 mentions)
Fos choline 12, by Anatrace (1 mentions)
Nupage novex bis tris gel, by Thermo Fisher Scientific (1 mentions)
Complete edta free protease inhibitor, by Roche (1 mentions)
N dodecylphosphocholine, by Anatrace (1 mentions)
Amicon ultracel 100 k, by Merck Group (1 mentions)
Histrap high performance column, by GE Healthcare (1 mentions)
High performance liquid chromatography (hplc), by Shimadzu (1 mentions)
Optilab t rex refractometer, by Wyatt Technology (1 mentions)
1260 infinity, by Agilent Technologies (1 mentions)
Akta fplc system, by GE Healthcare (1 mentions)
690 nm laser, by Wyatt Technology (1 mentions)
Amicon ultra centrifugal filter unit, by Merck Group (1 mentions)
27 protocols using superdex s200 10 300 gl column
1
SEC-MALS Analysis of FzCRD and Norrin–Fz4CRD Complex
2
Crystallization of GTD-Fab Complexes
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Equimolar amounts of GTD and Fab B2 or B1 were incubated on ice for 2.5 h and the resulting complexes purified by gel filtration on a Superdex S200 10/300 GL column (GE Healthcare Bio- Sciences, Pittsburgh PA, USA) equilibrated in 50 mM Tris pH 7.5, 150 mM NaCl. Peak fractions corresponding to the complex were pooled, concentrated and used in crystallization experiments.
All crystals were grown at 293 K. Crystals of the complex of GTD and FabB1 were generated by microseed-matrix screening (D'Arcy et al., 2007 (link)). Original crystals were grown by mixing equal amounts of protein complex at 20 mg/mL and reservoir solution consisting of 0.1 M HEPES pH 7.5, 40% polyethylene glycol 400 and 0.2 M calcium acetate. These crystals were resuspended in 50 μL of reservoir solution, crushed with a seed bead (Hampton Research, Aliso Viejo, CA), and used for seeding into drops consisting of equal amounts of protein complex at 10 mg/mL and reservoir consisting of 0.1 M sodium cacodylate pH 5.0, 40% PEG 300 and 0.2 M calcium acetate. Crystals were frozen directly in liquid nitrogen. Crystals of the complex of GTD and Fab B2 were grown by mixing equal amounts of protein complex at 15 mg/mL and reservoir consisting of 0.1 M TRIS pH 8.2 and 39% polyethylene glycol 400. Crystals were frozen directly in liquid nitrogen.
All crystals were grown at 293 K. Crystals of the complex of GTD and FabB1 were generated by microseed-matrix screening (D'Arcy et al., 2007 (link)). Original crystals were grown by mixing equal amounts of protein complex at 20 mg/mL and reservoir solution consisting of 0.1 M HEPES pH 7.5, 40% polyethylene glycol 400 and 0.2 M calcium acetate. These crystals were resuspended in 50 μL of reservoir solution, crushed with a seed bead (Hampton Research, Aliso Viejo, CA), and used for seeding into drops consisting of equal amounts of protein complex at 10 mg/mL and reservoir consisting of 0.1 M sodium cacodylate pH 5.0, 40% PEG 300 and 0.2 M calcium acetate. Crystals were frozen directly in liquid nitrogen. Crystals of the complex of GTD and Fab B2 were grown by mixing equal amounts of protein complex at 15 mg/mL and reservoir consisting of 0.1 M TRIS pH 8.2 and 39% polyethylene glycol 400. Crystals were frozen directly in liquid nitrogen.
3
Purification of Recombinant NTR 2.0 Protein
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The gene encoding NTR 2.0 was ordered pre-synthesised from Twist Bioscience (San Francisco, CA, USA). The gene fragment was cloned into pET28a(+) (Novagen) which expresses a His6 tag at the C-terminus. The expression construct was used to transform E. coli BL21 cells and grown in LB medium containing 50 µg·mL−1 kanamycin at 37 °C. After induction with 0.5 mM IPTG, the culture was incubated for a further 24 h at 18 °C. The culture was harvested by centrifugation at 2600× g for 20 min. NTR 2.0 was purified using nickel affinity chromatography where bound protein was eluted over a two-step wash and elute method, involving an initial wash at 60 mM imidazole to remove loosely bound non-target proteins and a second wash at 1 M imidazole to elute NTR 2.0. For further purification, size exclusion chromatography was performed using a Superdex S200 10/300 GL column (GE Healthcare, Pollards Wood, UK) connected to an ÄKTA BasicTM FPLC system and a 40 mM Tris-HCl pH 7.0, 200 mM NaCl buffer. Fractions containing the target protein were assessed for purity by SDS-PAGE analysis and quantified by their 280 nm absorbance trace.
4
Purification of Chloroplast RNA Editing Proteins
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Cell pellets containing rAtRIP2, rAtRIP9, rZmRIP9 and rAtOZ1 were resuspended in a chilled lysis buffer with 50 mM HEPES pH 7.0, 250 mM NaCl, 10% glycerol, 0.01% (w/v) CHAPS, and 10 mM imidazole. Similarly, cell pellets containing rAtDYW1 and AtORRM1 were resuspended in a chilled lysis buffer with 20 mM tris–HCl pH 7.3, 150 mM NaCl, 10% glycerol, 0.01% (w/v) CHAPS, and 10 mM imidazole. Cell pellets for rAtCRR4 were resuspended in 50 mM tris–HCl pH 7.3 @ RT, 250 mM NaCl, 1% glycerol, 0.01% (w/v) CHAPS, and 10 mM imidazole. PMSF suspended in 2-proponol was added to a final concentration of 1 mM before cell lysis. Cell suspensions were sonicated 6 times at 80% of the maximum amplitude for 20 s, each with 1 min of rest in between bursts. Insoluble cellular debris was removed through pelleting in the centrifuge at 12,000×g for 30 min. The N-terminal his-tagged proteins rAtDYW1, rAtRIP2, rAtRIP9, ZmRIP9, rAtORRM1, rAtCRR4, and rAtOZ1 were purified using IMAC with His-PURE NiNTA resin. Proteins were assessed for purity using Coomassie stained SDS-PAGE. Size exclusion chromatography was performed with a Superdex S200 10/300 GL column (GE Life Sciences). The column was equilibrated and AtCRR4, AtORRM1, AtRIP2m ZmRIP9 proteins were purified using a running buffer composed of 20 mM tris–HCl pH 7.3, 150 mM NaCl, 10% glycerol at a flow rate of 0.25 mL/min.
5
Purification of His-tagged Pf PLSCR Protein
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Recovered His::PfPLSCR protein was refolded by rapid dilution into IMAC buffer A [250 mM NaCl, 0.5 mM CaCl2, 1 mM TCEP, 0.1 % Fos-choline-12 (n-Dodecyl-phosphocholine, Anatrace), 25 mM HEPES pH 7.5], resulting in a final SDS concentration of 0.1 %. Refolded protein was filtered through a 0.45 μm filter prior to immobilised metal affinity chromatography (IMAC) using a HisTRAP™ High Performance column (GE Healthcare), which was equilibrated with IMAC buffer A.
The loaded protein column was washed with several column volumes of wash buffer [IMAC buffer A + 0.4 % SDS] and IMAC buffer A. The protein was eluted in a linear imidazole gradient using IMAC buffer B [IMAC buffer A + 1 M imidazole]. Fractions containing the desired protein were concentrated (Amicon Ultracel 100 K, Millipore) and further purified by size-exclusion chromatography (SEC) using a Superdex S200 10/300 G L column (GE Healthcare) in SEC buffer [250 mM NaCl, 0.5 mM CaCl2, 1 mM TCEP, 0.1 % FC-12, 25 mM HEPES pH 7.5]. All buffers contained cOmplete™ EDTA-free protease inhibitors (Roche). Purity of the protein was assessed by SDS-PAGE using NuPAGE™ Novex® Bis-Tris gels and blue native PAGE was performed by using the NativePAGE™ Novex® Bis-Tris Gel System and G250 additive (Life Technologies).
The loaded protein column was washed with several column volumes of wash buffer [IMAC buffer A + 0.4 % SDS] and IMAC buffer A. The protein was eluted in a linear imidazole gradient using IMAC buffer B [IMAC buffer A + 1 M imidazole]. Fractions containing the desired protein were concentrated (Amicon Ultracel 100 K, Millipore) and further purified by size-exclusion chromatography (SEC) using a Superdex S200 10/300 G L column (GE Healthcare) in SEC buffer [250 mM NaCl, 0.5 mM CaCl2, 1 mM TCEP, 0.1 % FC-12, 25 mM HEPES pH 7.5]. All buffers contained cOmplete™ EDTA-free protease inhibitors (Roche). Purity of the protein was assessed by SDS-PAGE using NuPAGE™ Novex® Bis-Tris gels and blue native PAGE was performed by using the NativePAGE™ Novex® Bis-Tris Gel System and G250 additive (Life Technologies).
6
Purification and Analysis of PKM2 Variants
7
MALS Characterization of SARAF Protein
8
Analytical Gel Filtration of Proteins
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Analytical gel filtration was carried out using a Superdex S200 10/300 Gl column (GE Healthcare) in 20 mM Hepes-KOH pH 7.5 and 200 mM sodium chloride, unless otherwise stated. 0.5 ml fractions were collected encompassing the void (7 ml) and bed (21 ml) volumes of the column. Fractions were separated by SDS-PAGE and stained with Coomassie Brilliant Blue (SERVA).
9
Size Exclusion Chromatography of Protein Variants
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SEC was performed with a Superdex S200 10/300 GL column (GE Healthcare) equilibrated in a buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM β-mercaptoethanol and 5 mM EDTA. Separations were performed at 20°C with a flow rate of 0.5 ml/min by HPLC (Agilent Technologies 1260 infinity). The samples (50 μl) were injected at a concentration of 2.5 mg/ml. On-line MALLS detection was performed with a miniDawn Treos detector (Wyatt Technology Corp., Santa Barbara, CA) using a laser emitting at 690 nm and by refractive index measurement using a Shodex RI-101 (Shodex). Data analyses were performed as described previously (Gerard et al., 2007 ). The S200 Superdex column was calibrated with proteins of known Stokes’ radii (RS), according to the method described by (Uversky, 1993 ), in order to extrapolate the relative RS of WT and NEECK mutant.
10
SEC-MALS Analysis of CC3ext
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SEC was performed on a Superdex S200 10/300 GL column linked to an AKTA FPLC system (GE Healthcare) at 4 °C. MALS measurements were performed in-line with the SEC using a three-angle (i.e. 45°, 90° and 135°) miniDawn light-scattering instrument equipped with a 690 nm laser (Wyatt Technologies); however, due to elution of the CC3ext in the void volume of the column, accurate molecular weight estimates could not be derived.
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