Superdex 200 10 300 column

Manufactured by Cytiva

Sourced in Sweden

The Superdex 200 10/300 column is a size exclusion chromatography column designed for the separation and purification of biomolecules such as proteins, peptides, and nucleic acids. The column has a bed volume of 24 mL and a separation range suitable for the analysis of molecules with molecular weights between 10,000 and 600,000 Da.

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Lab products found in correlation

Histrap excel column, by Cytiva (2 mentions) Expi293f cells, by Thermo Fisher Scientific (2 mentions) Expifectamine 293 transfection kit, by Thermo Fisher Scientific (2 mentions) Dawn 8, by Wyatt Technology (2 mentions) Astra 6, by Wyatt Technology (2 mentions) Vitrobot, by Thermo Fisher Scientific (2 mentions) Dawn heleos 2 detector, by Wyatt Technology (1 mentions) Optilab t rex differential refractive index detector, by Wyatt Technology (1 mentions) Kta purifier, by Cytiva (1 mentions) Prism 9, by GraphPad (1 mentions) Grace s insect medium, by Thermo Fisher Scientific (1 mentions) Fugene hd transfection reagent, by Promega (1 mentions) Minidawn treos, by Wyatt Technology (1 mentions) 1260 infinity hplc, by Agilent Technologies (1 mentions) λ phosphatase, by New England Biolabs (1 mentions) Apyrase, by Merck Group (1 mentions) Agilent 1260 uv, by Agilent Technologies (1 mentions) Optilab t rex, by Wyatt Technology (1 mentions) Histrap column, by Cytiva (1 mentions)

Spelling variants of this product

Superdex 200 (66 citations) Superdex 200 column (50 citations) Superdex 200 10/300 (14 citations)

15 protocols using superdex 200 10 300 column

Cryo-EM sample preparation of BoNT/B and BoNT/E

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BoNT/B was prepared with an additional size exclusion chromatography step using a Superdex200 10/300 column (Cytiva, Uppsala, Sweden) pre-equilibrated in 20 mM HEPES pH 7.5, 200 mM NaCl, 0.5 mM TCEP. Sample at 0.05 mg/mL in 20 mM HEPES pH 7.5, 50 mM NaCl was pipetted onto glow-discharged holey carbon cryo-EM grids (Quantifoil Cu R0.6/1) and frozen in liquid ethane using a Vitrobot (Thermo Fisher Scientific, Göteborg, Sweden).
BoNT/E was prepared with an additional size exclusion chromatography step using a Superdex200 10/300 column (Cytiva, Uppsala, Sweden) pre-equilibrated in 20 mM HEPES pH 7.2, 200 mM NaCl, 1 mM TCEP. Sample at 0.1 mg/mL in 20 mM HEPES pH 7.5, 50 mM NaCl was pipetted onto glow-discharged holey carbon cryo-EM grids (Cu R1.2/1.3, Quantifoil, Großlöbichau, Germany) and frozen in liquid ethane using a Vitrobot (Thermo Fisher Scientific, Göteborg, Sweden).

Košenina S., Martínez-Carranza M., Davies J.R., Masuyer G, & Stenmark P. (2021). Structural Analysis of Botulinum Neurotoxins Type B and E by Cryo-EM. Toxins, 14(1), 14.

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Size-Exclusion Chromatography of Protein-DNA Complexes

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We injected 100 μl samples of Gcf1p, Gcf1p/DNA20, Gcf1p/DNA50 or Gcf1p_CC/DNA50 (Supplementary Table S3) at a protein concentration of 2 mg/ml onto a Superdex 200 10/300 column (Cytiva). Complexes were prepared at protein:DNA ratios of 1:1.2 (Gcf1p/DNA20) and 2:1.2 (Gcf1p/DNA50 and Gcf1p_CC/DNA50). Column equilibration and running buffer for isolated Gcf1p was 750 mM NaCl, 50 mM Tris–HCl pH 8.0, and 20 mM NaCl, 50 mM Tris–HCl pH 8.0 for the complexes. For both cases the flow rate was 0.5 ml/min at RT. The column was coupled to a MALLS system including a DAWN-HELEOS-II detector (Wyatt Technology) with a laser emission wavelength of 664.3 nm. Peak concentrations were measured with an Optilab T-rEX differential refractive index detector (Wyatt Technology) assuming a dn/dC of 0.185 ml/g. Molecular weights (MWs) and complex ratios were determined using conjugate calculations in ASTRA 6.0.5.3 (Wyatt Technology). We also measured absorbance at 280nm. Experiments were carried out at the Automated Crystallography Platform at the Barcelona Science Park.

Tarrés-Solé A., Battistini F., Gerhold J.M., Piétrement O., Martínez-García B., Ruiz-López E., Lyonnais S., Bernadó P., Roca J., Orozco M., Le Cam E., Sedman J, & Solà M. (2023). Structural analysis of the Candida albicans mitochondrial DNA maintenance factor Gcf1p reveals a dynamic DNA-bridging mechanism. Nucleic Acids Research, 51(11), 5864-5882.

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Size Exclusion Chromatography of LrsL

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Size exclusion chromatography combined with multiangle light scattering was performed on a Superdex 200 10/300 column (Cytiva) on an Agilent HPLC setup. 5.5 mg/ml of LrsL were injected in the column and analyzed in 20 mM HEPES, pH 7.5, 200 mM NaCl, 1 mM TCEP, and 0.01% of NaN₃. The molecular weight estimation and refractive index were calculated using Astra Multiangle Light Scattering software (Wyatt Technology).

Rehman Z.U., Momin A.A., Aldehaiman A., Irum T., Grünberg R, & Arold S.T. (2022). The exceptionally efficient quorum quenching enzyme LrsL suppresses Pseudomonas aeruginosa biofilm production. Frontiers in Microbiology, 13, 977673.

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Recombinant anti-CD3ε single-chain Fv

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A synthetic gene encoding an anti-human CD3ε monoclonal antibody (clone UCHT1) in single-chain Fv format was synthesized (Integrated DNA Technologies). To construct expression vectors encoding scDbs, genes encoding the variable domains of heavy and light chains of the HapImmune™ antibodies and UCHT1 with a His-tag at the C-terminus were cloned into the mammalian expression vector pBCAG. Expi293F cells (Thermo Fisher) were transiently transfected with expression vectors using the ExpiFectamine 293 Transfection Kit (Thermo Fisher), according to the manufacturer’s protocol. Transfected cells were incubated at 37°C with 8% CO₂ for 7 days, and scDbs were purified from supernatants using a HisTrap excel column (Cytiva) followed by size exclusion chromatography using a Superdex 200 10/300 column (Cytiva). The purity of the scDb proteins was analyzed by SDS-PAGE.

Hattori T., Maso L., Araki K.Y., Koide A., Hayman J., Akkapeddi P., Bang I., Neel B.G, & Koide S. (2022). Creating MHC-Restricted Neoantigens with Covalent Inhibitors That Can Be Targeted by Immune Therapy. Cancer Discovery, 13(1), 132-145.

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SEC-MALS Analysis of TPR-like Domain

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A total of 250 μl of purified TPR-like domain at 1–3 mg/ml were fractionated by SEC using a Superdex 200 10/300 column (Cytiva) equilibrated in 20 mM Tris-HCl pH 6.8, 0.2 M NaCl, 5% glycerol, and 1 mM DTT and an ÄKTA purifier (Cytiva) at a flow rate of 0.5 ml/min. The eluted samples were characterized by measuring the refractive index and multi-angle light scattering (MALS), using Optilab T-rEX and DAWN 8⁺ (Wyatt). Data were analyzed using the Astra6 software (Wyatt) to obtain the molar mass of each protein and represented using GraphPad Prism 9.

Francisco-Velilla R., Embarc-Buh A., del Caño-Ochoa F., Abellan S., Vilar M., Alvarez S., Fernandez-Jaen A., Kour S., Rajan D.S., Pandey U.B., Ramón-Maiques S, & Martinez-Salas E. (2022). Functional and structural deficiencies of Gemin5 variants associated with neurological disorders. Life Science Alliance, 5(7), e202201403.

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CDK2-cyclinA-p27 Complex Assembly

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T160pCDK2-cyclinA-p27 (5.3 mg) in 40 mM Tris–HCl (pH 7.6), 1 mM ATP, 10 mM MgCl₂ buffer was incubated with 4.2 mg of T160pCDK2-cyclin A in a total volume of 2 mL at 25 °C for 2 h. The reaction mix was loaded on to a Superdex 200 16/60 HR column (Cytiva) equilibrated in 20 mM Tris pH 7.8, 300 mM NaCl, 0.5 mM TCEP buffer to remove the T160pCDK2-cyclin A. To assemble the complex, equimolar concentrations of purified CDK2-cyclinA-p27 (phosphorylated on CDK2 T160 and p27 T187) and SKP1-SKP2 complexes were incubated with a molar excess of CKS1 for 1 h at 4 °C and separated by SEC on a Superdex 200 10/300 column (Cytiva) equilibrated in 20 mM Tris pH 7.8, 300 mM NaCl, 0.5 mM TCEP.

Rowland R.J., Heath R., Maskell D., Thompson R.F., Ranson N.A., Blaza J.N., Endicott J.A., Noble M.E, & Salamina M. (2023). Cryo-EM structure of SKP1-SKP2-CKS1 in complex with CDK2-cyclin A-p27KIP1. Scientific Reports, 13, 10718.

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Synthetic scFv Anti-CD3ε Antibody Production

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A synthetic gene encoding an anti-human CD3ε monoclonal antibody (clone UCHT1) in an scFv format was synthesized (Integrated DNA Technologies). To construct expression vectors encoding scDbs, genes encoding the variable domains of heavy and light chains of the HapImmune antibodies and UCHT1 with a His-tag at the C-terminus were cloned into the mammalian expression vector pBCAG. Expi293F cells (Thermo Fisher) were transiently transfected with expression vectors using the ExpiFectamine 293 Transfection Kit (Thermo Fisher) according to the manufacturer's protocol. Transfected cells were incubated at 37°C with 8% CO₂ for 7 days, and scDbs were purified from supernatants using a HisTrap excel column (Cytiva), followed by size-exclusion chromatography using a Superdex 200 10/300 column (Cytiva). The purity of the scDb proteins was analyzed by SDS-PAGE.

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Expression and Purification of His-Tagged AGO2 Protein

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His-AGO2 protein was expressed in Spodoptera frugiperda (Sf9) insect cells using a baculovirus expression system. Sf9 cells were grown in Grace’s insect medium (Gibco) supplemented with 1% FBS to a density of 2 × 10⁶ cells per ml at 27°C in a shake flask, and then co-transfected pQB3-AGO2 plasmid and qBac-III (qBac Bacmid) with FuGENE HD Transfection Reagent (Promega) to generate recombinant baculoviruses which were collected at 4–5 days. The baculoviruses supernatants were amplified for multiple rounds with high titer and then used to infect new Sf9 cells at specific MOI for another 4–5 days. Cells were harvested by centrifugation and resuspended in a nondenaturing lysis buffer for ultrasonic lysis. The lysate was centrifugated, and the supernatant was incubated with Ni-NTA resin (P2229S, Beyotime) at 4°C overnight in a gently rotate speed. After several additional washes, the protein was eluted in elution buffer, and the eluted protein was further purified using a Superdex200 10/300 column (Cytiva). Purified His-AGO2 was concentrated in high salt buffer (20 mM HEPES, pH 7.5, 1 M NaCl, 2% glycerol, 1 mM DTT). Final proteins were flash frozen in liquid N₂ and stored at −80°C. All purification steps were performed on ice or at 4°C.

Wang Y.L., Feng L.L., Shi J., Chen W.Y., Bie S.Y., Bai S.M., Zeng G.D., Wang R.Z., Zheng J., Wan X.B, & Fan X.J. (2023). CiRS-7 Enhances the Liquid-liquid Phase Separation of miRISC and Promotes DNA Damage Repair. Nucleus, 14(1), 2293599.

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SEC-MALS Analysis of Recombinant PDK1

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The oligomeric state and polydispersity of purified recombinant proteins were assessed by SEC-MALS. 50 μl of PDK1^SKD S241A (3 mg/ml), PDK1^LKD S241A (5 mg/ml), PDK1^FL S241A (1.5 mg/ml), PDK1^PIF-SKD S241A (5.6 mg/ml), or PDK1^SKD-PIF S241A (4 mg/ml) was injected onto a Superdex 200 10/300 column (Cytiva) operated by a 1260 Infinity HPLC (Agilent Technologies). Light scattering of a 690 nm laser was detected by a MiniDawn Treos (Wyatt) and the refractive index was measured by a Shodex RI-101 (Shodex) detector. The runs were done in 50 mM Tris pH 7.5, 100 mM NaCl, 2% glycerol, 1 mM TCEP, 2 mM MgCl₂, ±1 mM ATP.

Levina A., Fleming K.D., Burke J.E, & Leonard T.A. (2022). Activation of the essential kinase PDK1 by phosphoinositide-driven trans-autophosphorylation. Nature Communications, 13, 1874.

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Purified OTR and Engineered G Protein Complex Formation

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Purified OTR and engineered heterotrimeric G protein (Gα_o/qGβ₁Gγ₂) were mixed in a molar ratio of 1:1.2 in complexation buffer (25 mM Hepes (pH 7.5), 100 mM KCl, 1 mM MgCl₂, 0.01% (w/v) LMNG, 0.001% (w/v) CHS, 100 μM OT, and 100 μm TCEP). After 30 min, apyrase (0.8 U/ml; MilliporeSigma) and λ-phosphatase (1,000 U/ml, New England Biolabs) were added to the mixture. After 2 h, purified scFv16 was added at 4-fold molar excess over receptor, and complex formation was allowed to proceed overnight at 4 °C. Stable complex was isolated by SEC on a Superdex 200 10/300 column (Cytiva) equilibrated with blotting buffer (25 mM Hepes (pH 7.5), 100 mM KCl, 1 mM MgCl₂, 100 μM OT, and 100 μm TCEP, 0.001% (w/v) LMNG, and 0.0001% (w/v) CHS). Corresponding peak fractions were concentrated to 1 mg/ml for EM studies, using a 100-kDa MWCO Vivaspin Turbo PES (Sartorius) concentrator.

Waltenspühl Y., Ehrenmann J., Vacca S., Thom C., Medalia O, & Plückthun A. (2022). Structural basis for the activation and ligand recognition of the human oxytocin receptor. Nature Communications, 13, 4153.

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