The PdMCD WT and variants (Q115A, T116A, and E117A) were initially purified as follows. Cells were lysed by ultrasonification, and the lysate was cleared by ultracentrifugation at 50,000 rcf for 45 min at 4 °C and subsequently filtered through a 0.45-μm filter. The cleared lysate was loaded onto a 1-ml HisTrap FF column (GE Healthcare), and unbound protein was removed with 20 ml of 20 mm Tris-HCl, pH 7.9, 500 mm NaCl, 75 mm imidazole. The protein was eluted in 20 mm Tris-HCl, pH 7.9, 500 mm NaCl, and 500 mm imidazole and subsequently desalted with a HiTrap 5-ml desalting (GE Healthcare) column in 20 mm Tris-HCl, pH 7.9, 200 mm NaCl. After the addition of 20% glycerol, the enzymes were concentrated to 10 mg ml−1 and incubated with 500 μm FAD for 1 h on ice. The enzymes were diluted to 2 mg ml−1 in 20 mm Tris-HCl, pH 7.9, 200 mm NaCl. 100 μl were injected onto a Superdex 200 Increase 10/300 GL size-exclusion column (GE Healthcare) with a flow rate of 0.7 ml min−1 and a running buffer of 20 mm Tris-HCl, pH 7.9, 200 mm NaCl. The size-exclusion calibration curve was obtained using the gel filtration standard (Bio-Rad).
Superdex 200 increase 10 300 gl size exclusion column
Manufactured by GE Healthcare
Sourced in United States
The Superdex 200 Increase 10/300 GL is a size-exclusion chromatography column manufactured by GE Healthcare. It is designed for the separation and purification of proteins, peptides, and other biomolecules based on their molecular size. The column has a bed volume of 24 mL and an inner diameter of 10 mm, making it suitable for a wide range of applications in the laboratory.
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Lab products found in correlation
Akt explorer 100, by Cytiva (2 mentions)
Astra software package, by Wyatt Technology (2 mentions)
Bio beads, by Bio-Rad (2 mentions)
Gel filtration standard, by Bio-Rad (1 mentions)
Histrap ff column, by GE Healthcare (1 mentions)
Sec mals 9, by Malvern Panalytical (1 mentions)
Ri detector ve3580, by Malvern Panalytical (1 mentions)
Expi293f cells, by Thermo Fisher Scientific (1 mentions)
Cobalt talon resin, by Takara Bio (1 mentions)
Quikchange mutagenesis, by Agilent Technologies (1 mentions)
Astra v5, by Wyatt Technology (1 mentions)
Ni nta agarose bead, by Qiagen (1 mentions)
Superdex 200 10 300 gl size exclusion column, by GE Healthcare (1 mentions)
18 protocols using superdex 200 increase 10 300 gl size exclusion column
1
Purification and Characterization of PdMCD Variants
2
Protein Size-Exclusion Chromatography
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500 μL of protein samples were loaded to a Superdex 200 Increase 10/300 GL size-exclusion column (GE Healthcare Life Sciences) equilibrated at room temperature in PBS and run at 0.75 mL/min flow rate. Eluted proteins were measured by Viscotek Sec-Mals 9 and Viscotek RI detector VE3580 (Malvern Panalytical).
3
Determining Molecular Weights of Proteins
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To determine the apparent molecular weight of native PhBS, the size-exclusion chromatography Superose 12 10/300 GL column was calibrated with following markers from Gel Filtration Markers Kit (Millipore Sigma): cytochrome c (12.4 kDa), carbonic anhydrase (29 kDa), bovine serum albumin (66 kDa), alcohol dehydrogenase (150 kDa), and β-amylase (200 kDa). To determine the apparent molecular weight of purified recombinant BS proteins, Superdex 200 Increase 10/300 GL size-exclusion column (GE Healthcare) was used with phosphate-buffered saline (PBS) for column equilibration and elution. The column was calibrated with following markers: bovine thyroglobulin (670 kDa), bovine gamma globulin (158 kDa), chicken ovalbumin (44 kDa), horse myoglobulin (17 kDa), and vitamin B-12 (1.4 kDa).
4
Recombinant SARS-CoV-2 Spike Protein Production
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Plasmids encoding the receptor binding domains (RBDs) were designed based on GenBank sequences MN975262.1 (SARS-CoV-2), ABD72970.1 (SARS-CoV), AGZ48828.1 (WIV-1), MN996532.2 (RaTG13), QJE50589.1 (SHC014), AAT98580.1 (HKU1), and AAT84362 (OC43). Constructs were codon optimized and synthesized by IDT. QuikChange Mutagenesis (Agilent) was used to insert glycosylation sites at SARS-CoV-2 RBD residues 501 and/or 475 as well as for RBD variant mutations, B.1.351 (K417N/E484K/N501Y) and B.1.1.7 (N501Y). SARS-CoV-2 spike contained a C-terminal foldon trimerization domain and HRV 3C-cleavable 6xHis and 2xStrep II tags (102 (link)). All proteins were transiently expressed in Expi293F cells (ThermoFisher). 5 to 7 days post-transfection, supernatants were harvested by centrifugation and further purified using immobilized metal affinity chromatography (IMAC) with cobalt-TALON resin (Takara) followed by Superdex 200 Increase 10/300 GL size exclusion column (GE Healthcare).
5
Determining Protein Molecular Weights by SLS
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Multi-angle static light scattering (SLS) was used to estimate molecular weights. Wild-type PduOC and PduOC-His18Ala (H18A) mutant were purified using a MiniQ 4.5/50 PE column (GE Healthcare) before SLS measurements. The proteins were diluted in 10 mM Tris-HCl (pH 7.5), loaded onto the column, and eluted using a 38-column-volume linear gradient from 0 to 1 M NaCl in 10 mM Tris-HCl (pH 7.5). The molecular weights were estimated by passing the proteins through a Superdex 200 increase 10/300 GL size exclusion column (GE Healthcare), equilibrated with 100 mM Tris-HCl (pH 8) and 150 mM NaCl, connected to a mini-DAWN TREOS multi-angle SLS detector (Wyatt Technology) and a Shodex RI-101 refractive index detector. The absolute molecular masses were determined based on the measured light scattering and refractive index using the Astra v. 5.3.4 software (Wyatt Technology).
6
Protein Sample Purification by SEC
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Protein samples at different concentrations were prepared in 7 M GdmCl and dialyzed against 20 mM sodium borate at pH 8.5 to eliminate potential non-specific aggregates produced by the freeze-dry procedure. For every run, 200 μl of sample were injected in a Superdex 200 Increase 10/300 GL size-exclusion column (from GE Healthcare). The chromatography run was performed using a flow of 0.5 ml/min of a buffer solution with 20 mM sodium borate at pH 8.5 to separate the species with different molecular mass.
7
Oligomerization Analysis of Vpu Proteins
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We used size exclusion chromatography (SEC) to assess the oligomerization state of the purified MBP-FL Vpu, MBP-VpuΔh2h3, and MBP-VpuΔh3 proteins, as described previously. A buffer composed of 50 mM sodium phosphate (NaPi) pH 7.4, 150 mM NaCl, 200 μM TCEP and 10% glycerol was used to equilibrate the column prior to protein sample injection. Then 500 μL of protein sample was injected into the column and eluted by running a SEC program with 0.5 ml/min flow rate. For these experiments, we used a Superdex™ 200 increase 10/300 GL size-exclusion column (GE Healthcare) plugged into an AKTÄ explorer 100 (Amersham Biosciences) protein purifier system.
8
Recombinant Pex5 Protein Purification
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FLAG-Pex5-His and His-Ub-Pex5-FLAG were expressed in E. coli BL21* cells in DYT. Cultures were grown shaking at 30 °C and were induced at ~OD600 = 0.6 by the addition of IPTG (Cf=0.3 mM), then incubated shaking for ~5 h at 30 °C. Cells were lysed by sonication and the 25,000×g supernatant was batch bound to Ni-NTA resin for 30 min. The protein-bound resin was washed with Ni_A buffer for ~40 CVs. Pex5 was eluted with 10 CVs of Ni_B buffer. Pex5 was then repeatedly flowed over anti-FLAG affinity resin 5–7 times, before being eluted with ~10 CVs of Ni_A buffer containing 0.15 mg mL−1 FLAG peptide. The Pex5 eluted from FLAG resin was concentrated and run on a Superdex 200 increase 10/300 GL size exclusion column (GE Life Sciences).
9
Sortase-Mediated Pex5 Purification
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For Pex5 constructs containing a Sortase cleavage site (LPETGG) just upstream of the C-terminal 6xHis tag, Pex5 (30 μM) were incubated with sortase (5 μM) and fluorescein-conjugated peptide GGGK-FAM peptide (50 μM, Elim BioPharm) for 2 h at 4 °C. The mixture was run back over fresh Ni-NTA resin to remove uncleaved protein, and the resulting flow-through was concentrated and run on a Superdex 200 increase 10/300 GL size exclusion column (GE Life Sciences).
10
Purification of PercevalHR Protein
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PercevalHR was purified in a manner similar to ArcA, ArcB, and ArcC1, except that different buffers were used. Ni2+-Sepharose resin was pre-equilibrated in 25 mM KPi buffer, pH 8.0 with 500 mM NaCl with 5% (v/v) glycerol (buffer E) plus 10 mM imidazole. The resin was washed with buffer E plus 25 mM imidazole and protein was eluted with buffer E plus 250 mM imidazole. The most concentrated fractions were run on a Superdex 200 Increase 10/300 GL size-exclusion column (GE Healthcare) in 10 mM NaPi, pH 7.4 with 150 mM NaCl and 5% (v/v) glycerol. Protein containing fractions (1–2 mg mL−1) were aliquoted in volumes of 50 µL, flash-frozen and stored at −80 °C.
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