Superdex 200

Manufactured by Cytiva

Sourced in United States, United Kingdom, Sweden

Superdex 200 is a size-exclusion chromatography medium designed for the separation and purification of biomolecules such as proteins, peptides, and other macromolecules. It is composed of a cross-linked agarose and dextran matrix, providing a high resolution and wide separation range.

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Lab products found in correlation

Superdex 75, by Cytiva (3 mentions) Gel chromatography calibration kit hmw, by GE Healthcare (2 mentions) Monoq column, by Cytiva (2 mentions) Hitrap q, by Cytiva (2 mentions) Hispur ni nta, by Thermo Fisher Scientific (2 mentions) Mabselect sure, by Cytiva (2 mentions) Amicon ultra 15, by Merck Group (2 mentions) Ni nta resin, by Qiagen (2 mentions) His 60, by Takara Bio (2 mentions) Complete edta free protease inhibitor tablet, by Roche (2 mentions) Superdex 200 increase column, by Cytiva (2 mentions) Expi293f cells, by Thermo Fisher Scientific (2 mentions) Protease inhibitor cocktail, by Promega (1 mentions) Gapdh antibody, by Merck Group (1 mentions) Pfx polymerase, by Thermo Fisher Scientific (1 mentions) Ni nta agarose resin, by Merck Group (1 mentions) F 12k medium, by American Type Culture Collection (1 mentions) Rpmi 1640 medium, by Merck Group (1 mentions) Pd 10 column, by GE Healthcare (1 mentions) Histrap hp column, by Cytiva (1 mentions)

Close spelling variants of this product

Superdex 200 10/300 GL column (59 citations) Superdex 200 Increase 5/150 GL (8 citations) Superdex 200 size exclusion column (8 citations) Superdex 200 Increase 10/300 GL gel filtration column (6 citations) HiLoad 26/600 Superdex 200 pg (6 citations) Superdex 200 16/600 pg column (4 citations) HiLoad 16/600 Superdex 200 prep grade column (4 citations) HiLoad 16/60 Superdex 200 (4 citations) Superdex-200 SEC column (3 citations) Superdex 200 increase 10/300 SEC column (3 citations)

66 protocols using superdex 200

Expression and Purification of CD19-HSA Fusion

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Antibodies and Fab were expressed in CHO cells (44 (link)). Capture from cell culture media was accomplished by affinity chromatography using either MabSelect SuRE (Cytiva Life Sciences) for antibodies or CaptureSelect KappaXL (Thermo Fisher Scientific) for Fab. Subsequent purification was done by size exclusion chromatography using Superdex 200 for antibodies and Superdex 75 for Fab (Cytiva Life Sciences).
Human CD19 ECD (amino acids 21–289) was fused to the N-terminus of C-terminally His-tagged human serum albumin (HSA) and expressed in the FreeStyle 293-F system (Thermo Fisher Scientific). Protein was captured from cell culture media by HisPur Ni-NTA (Thermo Fisher Scientific) and further purified by Superdex 200 (Cytiva Life Sciences). Purified CD19-HSA-His was biotinylated with EZ-Link Sulfo-NHS-LC biotin (Thermo Fisher Scientific).

Boyles J.S., Sadowski D., Potter S., Vukojicic A., Parker J., Chang W.Y., Ma Y.L., Chambers M.G., Nelson J., Barmettler B., Smith E.M., Kersjes K., Himes E.R., Lin C., Lucchesi J., Brahmbhatt J., Sina R., Martin J.A., Maestri E., Wiethoff C.M., Dyas G.L., Linnik M.D., Na S., Witcher D.R., Budelsky A, & Rubtsova K. (2023). A nondepleting anti-CD19 antibody impairs B cell function and inhibits autoimmune diseases. JCI Insight, 8(13), e166137.

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Purification of Recombinant Pol γA and Pol γB

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Proteins were prepared as previously described (13 (link)). Briefly, Pol γA WT, Exo-deficient [D198A/E200A (exo⁻)], and mutants [Pol γA R853A, Pol γA R853Q, Pol γA R853W, and Pol γA R853A (exo⁻)] were expressed in insect cells Sf9 and purified using TALON (Clontech) and Superdex 200 (Cytiva) column chromatography. Pol γB WT and a deletion mutant Pol γB-ΔI4 (deletion of amino acids 136 to 182) were expressed in E. coli Rosetta BL21 DE3 and purified using Nickel-NTA (Qiagen) and cation-exchange (Mono S) chromatography. For structural studies, purified Pol γA and Pol γB were mixed at a 1:2 molar ratio on ice for 10 min and purified using Superdex 200 (Cytiva) column chromatography. The protein purity was estimated to be >95% per SDS–polyacrylamide gel electrophoresis analysis.

Park J., Herrmann G.K., Roy A., Shumate C.K., Cisneros G.A, & Yin Y.W. (2024). An interaction network in the polymerase active site is a prerequisite for Watson-Crick base pairing in Pol γ. Science Advances, 10(21), eadl3214.

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Recombinant Protein Expression and Purification

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Expression and purification were performed in variations of established protocols as previously reported^{46 ,57 (link)}. In short, proteins were produced in E.coli BL21Star (DE3) or BL21 (DE3) cod+ and purified depending on the affinity tag. For in in vivo biotinylation on the Avi-tag the plasmid pBirA (Avidity Nanomedicines, La Jolla, CA) was cotransformed and the cell culture procedure was adapted according to Avidity’s in vivo biotinylation protocol.
His₆-SUMO tagged Hsp90 was isolated with affinity chromatography using a Ni-IMAC column (HisTrap HP), followed by SUMO cleavage with SenP protease and a second Ni-IMAC to separate the His-tag free protein from uncleaved proteins and the His₆-SUMO-tag. The protein was then applied to an anion exchange column (HiTrap Q, Cytiva) and finally to a size exclusion column (Superdex 200, Cytiva).
Strep-tagII and simple His₆-tag preparations were performed with a two-step protocol, starting with affinity chromatography using either a Strep-Tactin column (Strep-Tactin Superflow, IBA Lifesciences) or a Ni-IMAC (Cytiva/ Ni-NTA Agarose, Qiagen) followed by Size exclusion chromatography (Superdex 200, Cytiva).

Vollmar L., Schimpf J., Hermann B, & Hugel T. (2024). Cochaperones convey the energy of ATP hydrolysis for directional action of Hsp90. Nature Communications, 15, 569.

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Purification of the wt-TSEN complex from E.coli

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The wt-TSEN complex was purified from E.coli using a multi-cistronic expression vector 18 (link) . Briefly, following IPTG overexpression cells were harvested, resuspended in lysis buffer (50 mM Tris pH 8.0, 10 % glycerol, 300 mM NaCl, 5 mM MgCl 2 , 0.1% Triton-X-100) with the addition of a cOmplete EDTA-free protease inhibitor tablet (Roche) , and lysed by sonication. Clarified lysate was incubated with HIS-60 (Takara) resin for 30-60 minutes and eluted with 250 mM lysis buffer supplemented with 250 mM Imidazole. The complex was further purified over a Superdex-200 (Cytivia) with the following buffer: 50 mM Tris pH 8.0, 200 mM NaCl, 5% glycerol, 5 mM MgCl 2 . For BS3 crosslinking, the complex was purified over a Superdex-200 (Cytivia) with the following buffer: 50 mM HEPES pH 8.0, 200 mM NaCl, 5% glycerol, 5 mM MgCl 2 . For cryo-EM, the purified complex was subsequently incubated with tRNA-ARG-2'F (purchased HPLC purified from Horizon) and further purified over a Superdex-200 pre-equilibrated with 20 mM HEPES pH 8.0, 100 mM NaCl, 5 mM MgCl 2 , and 2% glycerol. Protein-tRNA fractions were pooled and concentrated and then diluted 1:1 with 20 mM HEPES pH 8.0, 100 mM NaCl, 5 mM MgCl 2 buffer.

Hayne C.K., Butay K.J.U., Stewart Z.D., Krahn J.M., Perera L., Williams J.G., Petrovitch R.M., Deterding L.J., Matera A.G., Borgnia M.J, & Stanley R.E. (2023). Structural basis for pre-tRNA recognition and processing by the human tRNA splicing endonuclease complex. Nature structural & molecular biology, 30(6).

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Purification of wt-TSEN complex from E. coli

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The wt-TSEN complex was purified from E.coli using a multi-cistronic expression vector^{18 (link)}. Briefly, following IPTG overexpression cells were harvested, resuspended in lysis buffer (50 mM Tris pH 8.0, 10 % glycerol, 300 mM NaCl, 5 mM MgCl₂, 0.1% Triton-X-100) with the addition of a cOmplete EDTA-free protease inhibitor tablet (Roche) , and lysed by sonication. Clarified lysate was incubated with HIS-60 (Takara) resin for 30–60 minutes and eluted with 250 mM lysis buffer supplemented with 250 mM Imidazole. The complex was further purified over a Superdex-200 (Cytivia) with the following buffer: 50 mM Tris pH 8.0, 200 mM NaCl, 5% glycerol, 5 mM MgCl₂. For BS3 crosslinking, the complex was purified over a Superdex-200 (Cytivia) with the following buffer: 50 mM HEPES pH 8.0, 200 mM NaCl, 5% glycerol, 5 mM MgCl₂. For cryo-EM, the purified complex was subsequently incubated with tRNA-ARG-2’F (purchased HPLC purified from Horizon) and further purified over a Superdex-200 pre-equilibrated with 20 mM HEPES pH 8.0, 100 mM NaCl, 5 mM MgCl₂, and 2% glycerol. Protein-tRNA fractions were pooled and concentrated and then diluted 1:1 with 20 mM HEPES pH 8.0, 100 mM NaCl, 5 mM MgCl₂ buffer.

Frost D.O., Boire D., Gingras G, & Ptito M. (2000). Surgically created neural pathways mediate visual pattern discrimination. Proceedings of the National Academy of Sciences of the United States of America, 97(20), 11068-11073.

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Apoptosis Regulation in Mammalian Cells

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Pfx polymerase was obtained from Invitrogen (Thermo Fisher Scientific, USA); Ni-NTA agarose resin, GAPDH antibodies and RPMI-1640 medium from Sigma ALDRICH (Inc. Sigma-Aldrich Corp, MO, USA); F-12 K medium from ATCC (ATTC, VA, USA); Applixchange-G25M from AppliChem (AppliChem GmbH, Darmstadt, Germany); Superdex-200 from Amersham (GE Healthcare Europe GmbH, Austria); anti-Bid antibodies from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); cytochrome c antibodies and Annexin V-FITC Apoptosis Detection kit I from Becton and Dickinson Bioscience (Becton, Dickinson and Company, New Jersey, USA); Protease Inhibitor Cocktail from Promega (Promega Corporation, USA).

Orzechowska E.J., Kozlowska E., Czubaty A., Kozlowski P., Staron K, & Trzcinska-Danielewicz J. (2014). Controlled delivery of BID protein fused with TAT peptide sensitizes cancer cells to apoptosis. BMC Cancer, 14, 771.

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Purification and Characterization of Agdc1-6hp

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Agdc1-6hp was purified by column chromatography on HisTrap FF (1 ml) Novagen, USA) in 20 mM Tris, 0.5 mM NaCl pH 7.9, and 5 mM imidazole as binding buffer and 20 mM Tris, 0.5 mM NaCl pH 7.9, and 1 M imidazole as elution buffer. Finally, the purified protein was desalted with PD10 columns (GE Health Care Europe GmbH, Germany).
The indicative molecular mass determination of native Agdc1-6hp was done by gel filtration using Superdex™ 200 (Amersham Biosciences, UK). The flow rate was 1 ml/min and fractions of about 1 ml were collected for 182 min (buffer: 50 mM Tris pH 8 + 0.15 M NaCl). A calibration curve was constructed using blue dextran, ferritin, catalase, bovine albumin, RNAse A and vitamin B12 as standards.
The K_m value for gallic acid and protocatechuic acid were determined as described in “Assay for determination of gallic acid activity.” All measurements were done in triplicate and Michaelis-Menten and Hanes plots were constructed.
The Agdc1p concentration was determined using a Coomassie stained SDS-PAGE for the calculation of k_cat (Kunze et al., 1998 (link)).

Meier A.K., Worch S., Böer E., Hartmann A., Mascher M., Marzec M., Scholz U., Riechen J., Baronian K., Schauer F., Bode R, & Kunze G. (2017). Agdc1p – a Gallic Acid Decarboxylase Involved in the Degradation of Tannic Acid in the Yeast Blastobotrys (Arxula) adeninivorans. Frontiers in Microbiology, 8, 1777.

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Yeast Cell Lysis and Protein Fractionation

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Yeast cell cultures in a volume of 50 ml were grown in YPD to an optical density of OD600 = 1.0. The cells were then harvested by centrifugation and the cell pellet was washed with ice-cold water. The cells were pelleted and re-suspended in 1000 μl of lysis buffer (50 mM HEPES-KOH at pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 1 tablet of the complete inhibitor cocktail supplied by Roche) and lysed with acid-washed glass beads for 15 minutes in a vortex on full output. Cell lysate was centrifuged (15 min at 13,000 rpm) at 4°C to remove cell debris. The gel filtration column (Superdex 200; Amersham Biosciences) was washed and equilibrated using cold PBS (4°C). Lysates were passed over the gel filtration column with a flow rate of 0.5 ml/min. Samples were collected every 0.5 ml per tube and analyzed by western blot.

Fang O., Hu X., Wang L., Jiang N., Yang J., Li B, & Luo Z. (2018). Amn1 governs post-mitotic cell separation in Saccharomyces cerevisiae. PLoS Genetics, 14(10), e1007691.

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Recombinant Protein Expression in E. coli

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E. coli strain
BL21(DE3) pLysS and the pET28a expression vector were obtained from
Novagen Inc. (Madison, WI). Amicon centrifugal filter devices (3000 M_r cutoff) were purchased from Millipore (Bedford,
MA). Q-Sepharose FF, SP-Sepharose FF, Superdex-200, and His-Trap HP
columns were obtained from Amersham Biosciences. Kanamycin, isopropyl
thiogalactopyranoside (IPTG), and 12-O-tetradecanoylphorbol-13-acetate
(PMA) were obtained from Sigma. Human α-thrombin (IIa), FXIa, pKLK, and plasmin were purchased from Haematologic Technologies
Inc. tPA (Alteplase) was purchased from Genentech (South San Francisco,
CA). uPA was obtained from Calbiochem, EMD Biosciences Inc. (San Diego,
CA). Glu–Plg was obtained from Enzyme Research Laboratories
(South Bend, IN). εACA (Amicar) was obtained from ICN Biomedicals
Inc. (Aurora, OH). Normal pooled plasma (NPP) was purchased from George
King Bio-Medical Inc. (Overland Park, KS). Plasmin substrate S-2251
(H-d-Val-Leu-Lys-p-nitroanilide) and pKLK and FXIa substrate S-2366 (pyroGlu-Pro-Arg-p-nitroanilide) were obtained from Diapharma Inc. All other
reagents were of the highest purity commercially available.

Kumar Y., Vadivel K., Schmidt A.E., Ogueli G.I., Ponnuraj S.M., Rannulu N., Loo J.A., Bajaj M.S, & Bajaj S.P. (2014). Decoy Plasminogen Receptor Containing a Selective Kunitz-Inhibitory Domain. Biochemistry, 53(3), 505-517.

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Protein Purification by Gel Filtration

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The gel filtration column (Superdex 200; Amersham Biosciences) was washed and equilibrated by cold PBS (4 °C). Proteins were passed over the gel filtration column. The flow speed rate was 0.4 mL min⁻¹. Fractions were collected every 0.5 mL per tube and analyzed by western blot. Molecular mass was determined by Gel Filtration Calibration Kit HMW (GE Healthcare).

Li Y., Yao C.F., Xu F.J., Qu Y.Y., Li J.T., Lin Y., Cao Z.L., Lin P.C., Xu W., Zhao S.M, & Zhao J.Y. (2019). APC/CCDH1 synchronizes ribose-5-phosphate levels and DNA synthesis to cell cycle progression. Nature Communications, 10, 2502.

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