Superdex 200 increase size exclusion column

Manufactured by GE Healthcare

The Superdex 200 Increase size exclusion column is a laboratory equipment designed for the separation and purification of biomolecules based on their size and molecular weight. It is a versatile tool used in various applications such as protein purification, enzyme analysis, and sample preparation.

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Lab products found in correlation

Steritop filter, by Merck Group (2 mentions) Histrap ni nta column, by GE Healthcare (2 mentions) Multitron pro shaker, by Infors (2 mentions) Fectopro, by Polyplus Transfection (2 mentions) Total brain lipids, by GE Healthcare (2 mentions) Amicon ultra, by Merck Group (2 mentions) Vivaspin 2, by Sartorius (2 mentions) Nanodrop 2000, by Thermo Fisher Scientific (2 mentions) Endo h, by New England Biolabs (1 mentions) In fusion hd cloning kit, by Takara Bio (1 mentions) Hitrap desalting, by GE Healthcare (1 mentions) Kappaselect affinity column, by GE Healthcare (1 mentions) Hek293f cell, by Thermo Fisher Scientific (1 mentions) Geneart, by Thermo Fisher Scientific (1 mentions) Bl21 de3, by New England Biolabs (1 mentions) Ni nta, by Qiagen (1 mentions) Vivaspin, by GE Healthcare (1 mentions) Kta pure 25 system, by GE Healthcare (1 mentions) Freestyle medium, by Thermo Fisher Scientific (1 mentions)

13 protocols using superdex 200 increase size exclusion column

Production and Purification of Anti-CD22 Fab

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CD22_d6–d7 was transiently cotransfected with the heavy chain and light chain of the m971 Fab into HEK 293S cells at a 1:1.5:1 ratio of CD22:HC:LC DNA and using FectoPRO (Polyplus Transfection). Cells were cotransfected at a cell density of 0.8 × 10⁶ cells ml⁻¹ and incubated at 37 °C, 125 rpm, 8% CO₂ in a Multitron Pro shaker (Infors HT) for 5 to 7 days. After harvesting the cells by centrifugation at 6371g for 20 min, supernatants were retained and filtered using a 0.22 μm Steritop filter (EMD Millipore). Supernatants were passed through a HisTrap Ni-NTA column (GE Healthcare) at 4 ml min⁻¹. The column was washed with 20 mM Tris, pH 8.0, 150 mM NaCl, and 5 mM imidazole buffer before elution with an increasing gradient of imidazole (up to 500 mM). Fractions containing protein were pooled, concentrated, and separated on a Superdex 200 Increase size exclusion column (GE Healthcare) at 0.5 ml min⁻¹ in 20 mM Tris, pH 8.0, 150 mM NaCl buffer to achieve size homogeneity. To obtain deglycosylated samples, purified CD22_d6–d7–m971 Fab complex was treated with the enzyme Endo H (New England Biolabs) for 1 h at 37 °C. The deglycosylated complex was purified further via a second Superdex 200 Increase size-exclusion column (GE Healthcare) at 0.5 ml min⁻¹ in 20 mM Tris, pH 8.0, and 150 mM NaCl buffer.

Ereño-Orbea J., Liu X., Sicard T., Kucharska I., Li W., Borovsky D., Cui H., Feng Y., Dimitrov D.S, & Julien J.P. (2021). Structural details of monoclonal antibody m971 recognition of the membrane-proximal domain of CD22. The Journal of Biological Chemistry, 297(2), 100966.

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Fluorescence-based RNA Characterization

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Fluorescence was measured on a Photon Technologies International/820 Photomultiplier Detection System with excitation and emission tuned to 510 nm and 535 nm, respectively. Initial readings at 0 nm and 500 nM TO1-Biotin were performed in a 0.1 mm cuvette. Prefolded RNA was added to the same cuvette to a final concentration of 2 μM, and allowed to incubate at RT for approximately 5 minutes. The fluorescence signals were acquired and averaged over 60s. RNA samples (RNA 3,6, and 9-21) in 20 mM MOPS pH 7.0, 150 mM KCl, 10 μM EDTA at 20 μM concentration, and equimolar concentration of DFHBI-1T, YO3-Biotin, or both, were heated to 95°C for 3 minutes and incubated at RT for 20 minutes. Samples were run on an ÄKTApurifier at 1 ml/minute over a Superdex 200 Increase size exclusion column (GE Life Sciences) at room temperature. The running buffer was the same as the RNA folding buffer supplemented with 400 nM DFHBI-1T, 20 nM YO3-Biotin, or both. Fractions corresponding to the complex of interest were collected, and concentrations were immediately determined on a NanoDrop 2000 (Thermo Scientific). Fluorescence was measured on a Photon Technologies International/820 Photomultiplier Detection System. Measurements were performed at an excitation of either 469 nm or 580 nm, and an emission of 616 nm.

Trachman RJ I.I.I., Cojocaru R., Wu D., Piszczek G., Ryckelynck M., Unrau P.J, & Ferré-D’Amaré A.R. (2020). Structure-guided engineering of the homodimeric Mango-IV fluorescence turn-on aptamer yields an RNA FRET pair. Structure (London, England : 1993), 28(7), 776-785.e3.

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Protein Expression and Purification of LysRS

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PfLysRS (77–583) wabs constructed in the vector pET20b with a C-terminal 6× His tag. The protein was expressed in BL21 (DE3) strain with 0.2 mM isopropyl-beta-d-thiogalactopyranoside (IPTG) for 20 h at 16°C. The cell pellet (from 4 to 8 l) was lysed in the wash buffer containing 500 mM NaCl, 20 mM Tris–HCl pH 8.0, and 15 mM imidazole. Then, the supernatant of the lysate was loaded onto a Ni-HiTrap column and washed with wash buffer. The protein was eluted with the elution buffer containing 500 mM NaCl, 20 mM Tris–HCl pH 8.0, and 250 mM imidazole. Then the protein was concentrated and further purified by gel filtration, using a Superdex 200 Increase size exclusion column (GE Healthcare) with running buffer containing 20 mM HEPES pH 7.5, and 150 mM NaCl (Supplementary Figure S1). The peak fractions were then concentrated for thermal shift assays, surface plasmon resonance assays, ATP hydrolysis assays or crystallization experiments. HsLysRS (70–581) was constructed in the vector pET20b with a C-terminal 6× His tag, and purified similarly. The protein concentrations used in all the experiments were determined by the Bradford method (29 (link)).

Zhou J., Huang Z., Zheng L., Hei Z., Wang Z., Yu B., Jiang L., Wang J, & Fang P. (2020). Inhibition of Plasmodium falciparum Lysyl-tRNA synthetase via an anaplastic lymphoma kinase inhibitor. Nucleic Acids Research, 48(20), 11566-11576.

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Oligomerization and Ligand Binding Monitoring

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To monitor oligomerization and response to ligand binding, 10 μM RNA samples were refolded in 20 mM Mops (pH 7.0), 100 mM KCl, and 10 μM EDTA in the presence or the absence of equimolar chelerythrine by heating to 95 °C for 3 min and cooling at −1 °C/min to a final temperature of 25 °C. After thermal anneal, 5 mM of MgCl₂ was added and samples were incubated at room temperature for 10 min. Samples were run on an ÄKTApurifier at 1 ml/min over a Superdex 200 Increase size-exclusion column (GE Life Sciences) at room temperature using a running buffer composed of 20 mM Mops (pH 7.0), 100 mM KCl, 10 μM EDTA, and 5 mM MgCl₂. Analyses containing ligand were supplemented with 60 nM chelerythrine. Absorbance was monitored at 260, 280, and 295 nm.

Trachman RJ I.I.I., Passalacqua L.F, & Ferré-D’Amaré A.R. (2022). The bacterial yjdF riboswitch regulates translation through its tRNA-like fold. The Journal of Biological Chemistry, 298(6), 101934.

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Production and Purification of CD22d6-d7

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Full-length CD22 ECD (CD22_d1–d7, residues 20–687) was designed, expressed, and purified as previously described (11 (link)). The CD22_d6–d7 (residues 505–688) construct was generated from CD22_d1–d7 using In-Fusion HD Cloning Kit (Takara) following the manufacturer’s instructions. The construct was cloned into the pHLsec vector (34 (link)) using restriction enzymes AgeI and KpnI, such that a 6× His tag was added at the C terminus of the construct to facilitate affinity purification. CD22_d6–d7 was transiently transfected using the transfection reagent FectoPRO (Polyplus Transfection) in HEK 293 Gnt I^−/− (HEK 293S; ATCC CRL-3022) suspension cells for protein expression. HEK 293S cells were incubated at 37 °C, 180 rpm, 8% CO₂ in a Multitron Pro shaker (Infors HT) for 6 to 7 days. After harvesting the cells by centrifugation, supernatants were retained and filtered using a 0.22 μm Steritop filter (EMD Millipore). Supernatants were passed through a HisTrap Ni-NTA column (GE Healthcare) at 4 ml min⁻¹. The column was washed with 20 mM Tris, pH 8.0, 150 mM NaCl, and 5 mM imidazole buffer before elution with an increasing gradient of imidazole (up to 500 mM). Fractions containing CD22_d7–d6 were pooled, concentrated, and separated on a Superdex 200 Increase size exclusion column (GE Healthcare) at 0.5 ml min⁻¹ in 20 mM Tris, pH 8.0, 150 mM NaCl buffer to achieve size homogeneity.

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Engineered IL-2 Variant Cytokine Production

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Structure based designs were evaluated through analysis of the 2B5I with IL-2 cytokine receptor complex (CD25, IL2RB, IL2RG, and IL-2) using Pymol (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC). Nucleotide changes were introduced using the Geneious software suite. gblock fragments from IDT-DNA were cloned into a mammalian expression vector using Golden Gate cloning system (ThermoFisher). Constructs encoding effector Functionless Fc fusions (64 (link)) of IL-2 variants were stably integrated into CHO K1 cells and expressed at 32°C in a 6-day batch production. The proteins were purified from clarified culture media using MabSelect SuRe affinity column (GE Healthcare) followed by HiTrap Desalting (GE Healthcare), and final purification was performed on a Superdex 200 Increase size exclusion column (GE Healthcare). Proteins were stored at 0.5–3 mg/ml in 10 nM Acetate pH 5.2, 100 M NaCl for biophysical and functional assays.

Ghelani A., Bates D., Conner K., Wu M.Z., Lu J., Hu Y.L., Li C.M., Chaudhry A, & Sohn S.J. (2020). Defining the Threshold IL-2 Signal Required for Induction of Selective Treg Cell Responses Using Engineered IL-2 Muteins. Frontiers in Immunology, 11, 1106.

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Fab Protein Expression and Purification

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The Fab heavy and light chains of STIM003 and prezalumab were cloned into custom pcDNA3.4 expression vectors at GeneArt (Life Technologies). Fabs were transiently expressed in 200 mL HEK 293F cells (ThermoFisher Scientific) by co-transfecting 90 μg of the LC and the HC in a 1:2 ratio with FectoPRO (Polyplus Transfections). Purification of the Fabs was performed using a KappaSelect affinity column (GE Healthcare) and 100 mM glycine pH 2.2 as the elution buffer. Eluted fractions were immediately neutralized with 1 M Tris-HCl pH 9.0 and further purified using a MonoS ion exchange column and a Superdex 200 Increase size exclusion column (GE Healthcare).

Rujas E., Cui H., Sicard T., Semesi A, & Julien J.P. (2020). Structural characterization of the ICOS/ICOS-L immune complex reveals high molecular mimicry by therapeutic antibodies. Nature Communications, 11, 5066.

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Overexpression and Purification of ClpP Proteases

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Two different E. coli cell strains were used for overexpression. EcClpP was overexpressed in BLR (DE3) from Novagen and BsClpP in BL21 (DE3) from New England Biolabs. Overexpression and purification conditions for both proteins were similar. Cell strains transformed with the relevant plasmids were grown at 37 °C to an OD₆₀₀ of 0.7 followed by induction with 1 mM isopropyl β-d-thiogalactopyranoside and overnight overexpression at 25 °C. Cell pellets were resuspended in 20 mM Tris and 150 mM NaCl (pH 8.0) and lysed using an Avestin C3 Emulsiflex. Lysates were clarified by centrifugation at 12 000 rpm for 30 min and incubated with Ni-NTA (Qiagen) resin pre-equilibrated with 20 mM Tris and 150 mM NaCl (pH 8.0). Protein was eluted from Ni-NTA resin with 20 mM Tris, 150 mM NaCl, and 500 mM imidazole (pH 8.0) and concentrated. Gel filtration, using a Superdex 200 Increase size exclusion column (GE Healthcare), was used as a final purification step for both proteins. Gel filtration buffer conditions were 25 mM Tris, 100 mM KCl, and 10% glycerol (pH 7.5)^{58 (link)} for EcClpP and 25 mM HEPES, 100 mM KCl, 20 mM MgCl₂, 1 mM EDTA, and 10% glycerol (pH 7.6)^{19 (link)} for BsClpP. Protein concentrations were determined using the calculated molar extinction coefficient in 6 M guanidinium hydrochloride.^{59 (link)}

Lavey N.P., Coker J.A., Ruben E.A, & Duerfeldt A.S. (2016). Sclerotiamide: The First Non-Peptide-Based Natural Product Activator of Bacterial Caseinolytic Protease P. Journal of natural products, 79(4), 1193-1197.

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Purification and Crystallization of ICMT

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ICMT was purified as described above except that 1 mg/ml total brain lipids (Avanti) were added to the purification buffers. The elution from the antibody affinity column was concentrated to 500 µl using a 50 kDa concentrator (Amicon Ultra; Millipore) and applied to a Superdex 200 Increase size exclusion column (GE Healthcare) that was equilibrated in 10 mM Tris-HCl, pH 7.5, 150 mM KCl, 5 mM TCEP, 2 mM CaCl₂, 25 µM AdoHcy, 1 mM DMNG, and 0.02 mg/ml total brain lipids. Fractions containing ICMT were pooled, supplemented with 500 µM AdoHcy, and concentrated to 5–10 mg/ml using a 50 kDa concentrator (Vivaspin-2; Sartorius). ICMT crystals were obtained by vapor diffusion (using 300 nl of ICMT and 300 nl of crystallization solution and a Mosquito crystallization robot, TTP Labtech) over reservoirs containing 100 µl of precipitant solution. The crystals were obtained from 24–28% PEG400 (v/v), 200 mM CaCl₂ or 200 mM MgCl₂, 50 mM Na acetate, pH 5.0–6.5, at 4°C and reached a size of ~100–400 µm within 2–3 weeks. The crystals were then transferred through a series of five steps using the components of an equilibrated drop to increase the PEG 400 concentration to 35% before flash-cooling in liquid nitrogen.

Diver M.M., Pedi L., Koide A., Koide S, & Long S.B. (2018). Atomic structure of the eukaryotic intramembrane Ras methyltransferase ICMT. Nature, 553(7689), 526-529.

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Purification and Crystallization of ICMT

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Diver M.M., Pedi L., Koide A., Koide S, & Long S.B. (2018). Atomic structure of the eukaryotic intramembrane Ras methyltransferase ICMT. Nature, 553(7689), 526-529.

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