Superose 6 increase 10 300 gel filtration column

Manufactured by GE Healthcare

The Superose 6 Increase 10/300 gel filtration column is a laboratory equipment used for size-exclusion chromatography. It is designed to separate and purify molecules based on their size and molecular weight, allowing for the efficient fractionation of complex mixtures.

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Lab products found in correlation

Superdex 200 column, by GE Healthcare (1 mentions) Ni nta affinity column, by GE Healthcare (1 mentions) Polyethylenimine (pei), by Polysciences (1 mentions) Rbdomicron, by Sino Biological (1 mentions) Superose 6 10 300 gl column, by GE Healthcare (1 mentions) Vivaspin 20 concentrator, by Sartorius (1 mentions) Streptactin superflow plus columns, by Qiagen (1 mentions) Amicon ultra 4 centrifugal filter, by Merck Group (1 mentions)

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4 protocols using superose 6 increase 10 300 gel filtration column

Recombinant SARS-CoV-2 Spike RBD and Omicron Protein Production

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RBD_WT, RBD_Alpha, RBD_Beta, RBD_Gamma, RBD_Kappa, and RBD_Delta (subscript indicating the variant it derives from) of spike protein (residue 319–541) were cloned into pcDNA3.1 vector with an IL2 signal peptide at the N-terminal and a C-terminal 8 × His-tag. Plasmids were transfected into HEK293F cells (cell density between 1 and 1.5 million cells mL⁻¹) with polyethylenimine (Polysciences). The conditioned media were collected after four days, and the proteins were purified using a Ni-NTA affinity column (GE Life Sciences), and Superdex 200 column (GE Life Sciences) in the final buffer (20 mM HEPES, pH 7.2, 150 mM NaCl). RBD_Omicron (40592-V08H121) was purchased from SinoBiological.
The spike_Omicron (subscript indicating the variant it derives from) expression plasmid encoding the spike ectodomain (residue 1–1208) with six stabilizing Pro substitutions (F817P, A892P, A899P, A942P, K986P, and V987P) and a ‘GSAS’ substitution at the furin cleavage site (residues 682–685) was constructed as previous described [19 (link)]. It was then transfected into the HEK293F cells when the density reached 1 million cell mL⁻¹ and expressed for four days. The protein was purified using the Ni-NTA resin, followed by a Superose 6 increase 10/300 gel filtration column (GE Life Science), and eluted with the final buffer.

Chen C., Song X., Yu Y., Wang X., Xu H., Ji W., Ma J., Zhao C., Feng S., Wang Y., Su X.D, & Wang W. (2023). Aptamer-based nanointerferometer enables amplification-free ultrasensitive detection and differentiation of SARS-CoV-2 variants. Analytica Chimica Acta, 1260, 341207.

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Gel-filtration Analysis of Rep68-DNA Complexes

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Gel-filtration studies were performed with the Rep68 concentration held constant at 30 μM and the DNA held constant at 10 μM. The gel-filtration buffer used was: 10 mM Na₂HPO₄ pH 7.0, 150 mM NaCl, 1 mM TCEP and all results were plotted using Origin (Origin labs). The experiments shown in Supplementary Figure S2 were conducted on a Superose 6 10/300 GL column (GE Healthcare) while the experiment shown in Figure 2 was conducted on a Superose 6 Increase 10/300 gel filtration column (GE Healthcare).

Santosh V., Musayev F.N., Jaiswal R., Zárate-Pérez F., Vandewinkel B., Dierckx C., Endicott M., Sharifi K., Dryden K., Henckaerts E, & Escalante C.R. (2020). The Cryo-EM structure of AAV2 Rep68 in complex with ssDNA reveals a malleable AAA+ machine that can switch between oligomeric states. Nucleic Acids Research, 48(22), 12983-12999.

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Purification of Reconstituted 20S Proteasome and 20S-PA200 Complex

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The purification strategy was the same for both reconstituted 20S proteasome and 20S-PA200 complexes. Briefly, thawed cells were resuspended in 3-4 times volume of buffer W (50 mM Tris pH 7.5, 150 mM NaCl, 5% (v/v) glycerol, 1 mM DTT, 1 mM EDTA), lysed by sonication and the lysate cleared by centrifugation at 48,400 g for 30 minutes. The clear lysate was then passed through a 5 μm filter and loaded onto tandem Streptactin Superflow Plus columns (QIAGEN), equilibrated in buffer W, and eluted in buffer E (50 mM Tris pH7.5, 150 mM NaCl, 5% (v/v) glycerol, 1 mM DTT, 1mM EDTA, 2.5 mM d-desthiobiotin). Proteasome containing fractions were pooled together and the TwinStrep-tag cleaved by overnight dialysis against buffer W containing TEV protease, at a ratio of 1:50 (TEV to protein). The sample was filtered again with a 0.4 μm filter and loaded onto Streptactin Superflow Plus columns, where the flow-through was collected. The protein was concentrated using 30 kDa cut-off Vivaspin 20 concentrators (Sartorius) and loaded onto a Superose 6 Increase 10/300 gel filtration column (GE Healthcare) equilibrated with 50 mM Tris pH 7.4, 100 mM NaCl, 1 mM EDTA (Figure S1B). Typical yields for the purification of reconstituted 20S proteasomes and 20S-PA200 complexes are 2-3 mg and 1 mg of protein purified from 1 L of Sf9 cultures, respectively.

Toste Rêgo A, & da Fonseca P.C. (2019). Characterization of Fully Recombinant Human 20S and 20S-PA200 Proteasome Complexes. Molecular Cell, 76(1), 138-147.e5.

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Structural Characterization of Rep68-DNA Complex

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The Rep68-dT₂₅ complex was prepared as previously described (23 (link)). In short, Rep68 was mixed with a 25-mer poly-dT oligonucleotide (dT₂₅), purified to homogeneity and concentrated in the presence of 0.05% n-octyl-β-d-glucopyranoside (OG). The Rep68-AAVS1-15 ssDNA complex was made by incubating 30μM protein with 15 μM ss AAVS1-15 DNA in reaction buffer (10 mM Na(PO₄)₂ pH 7.0, 150 mM NaCl 1 mM TCEP) for 15 min at room temperature. The complex was then concentrated to 200 μl using Amicon Ultra-4 centrifugal filter (Millipore) and loaded onto a pre-equilibrated Superose 6 Increase 10/300 gel filtration column (GE Healthcare). Eluted complex was concentrated back to 1/8th and 1/16th of original protein concentration. C-Flat carbon grids CF1.2/1.3-4C, 400 mesh Cu (Electron Microscopy Sciences CF413-50) were glow-discharged for 45 seconds with amylamine using a PELCO easiGlow™ glow-discharge system. Just before spotting the sample, 5 mM MgCl₂ and 5 mM ATP_γS (final concentration) was added to the complex and incubated at room temperature for 5 min. 3.5 μl sample was spotted, blotted and plunged frozen into liquid ethane using the Vitrobot unit and stored in liquid nitrogen.

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