Superdex 200 increase 10 300 column

Manufactured by GE Healthcare

Sourced in United States, Germany

The Superdex 200 Increase 10/300 column is a size exclusion chromatography column manufactured by GE Healthcare. It is designed for the separation and purification of biomolecules based on their molecular size. The column has a bed volume of 24 mL and is compatible with standard chromatography systems.

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Superdex 200 (746 citations) Superdex 200 column (565 citations) Superdex 200 10/300 (112 citations) Superdex 200 Increase (76 citations) Superdex 200 Increase 10/300 (71 citations) Superdex Increase 200 10/300 columns (2 citations)

173 protocols using superdex 200 increase 10 300 column

Expression and Purification of HIV-1 Trimer Proteins

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1-18 IgG was expressed by transient transfection in Expi293 cells (Thermo Fisher) and purified from transfected cell supernatants using a HiTrap MabSelect Protein A column (GE Life Sciences). Fab fragments were isolated as described (Diskin et al., 2011 (link)) after papain cleavage of 1-18 IgG, removal of Fc by protein A chromatography, and then purification by size exclusion chromatography (SEC) on a Superdex-200 Increase 10/300 column (GE Life Sciences) equilibrated with TBS (20 mM Tris pH 8.0, 150 mM NaCl). 1-55 Fab was expressed as a light-chain C-terminal His₆-tagged Fab by transient transfection in 293-6E cells (National Research Council of Canada) and purified from supernatants using Ni²⁺-NTA affinity chromatography (GE Life Sciences) followed by SEC purification with a Superdex-200 Increase 10/300 column equilibrated with TBS. All Fabs were stored at 4°C.
BG505_SOSIP.664 trimer was stably expressed in Chinese hamster ovary cells (kind gift of J.P. Moore and A. Cupo) as described (Chung et al., 2014 (link)) and purified from cell culture supernatant over a 2G12 immunoaffinity column followed by SEC purification on a Superdex-200 16/60 column (GE Life Sciences) equilibrated with TBS. RC1_SOSIP.664 was expressed by transient transfection in 293-6E cells and purified as described (Escolano et al., 2019 (link)). Individual SEC fractions of each SOSIP trimer were stored at 4°C.

Schommers P., Gruell H., Abernathy M.E., Tran M.K., Dingens A.S., Gristick H.B., Barnes C.O., Schoofs T., Schlotz M., Vanshylla K., Kreer C., Weiland D., Holtick U., Scheid C., Valter M.M., van Gils M.J., Sanders R.W., Vehreschild J.J., Cornely O.A., Lehmann C., Fätkenheuer G., Seaman M.S., Bloom J.D., Bjorkman P.J, & Klein F. (2020). Restriction of HIV-1 Escape by a Highly Broad and Potent Neutralizing Antibody. Cell, 180(3), 471-489.e22.

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Sortase A-mediated Ligation and Characterization of 1NOG-Antibody Complexes

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The expression and purification procedures of Sortase A and 1NOG-His trimer were described previously (38 (link)). Briefly, the proteins were produced in Rosetta2 E. coli cells (Novagen) and purified with Ni NTA-agarose beads (Qiagen) and Superdex 200 increase 10/300 column (GE Healthcare). For protein ligation reaction, GPCysRRLL-LPETG-Strep and GGGGGSGSP-1NOG-His trimer were mixed with a molar ratio of 3:1 in TBS buffer plus 2 mM CaCl₂ and 1 μM purified Sortase A. After incubation at room temperature for 50 minutes, 2 mM EGTA (final concentration) was added, and the reaction mixture was immediately purified with a Superdex 200 increase 10/300 column (GE Healthcare) to obtain GPCysRRLL-LPETG-1NOG trimer.
To make samples of GPCysRRLL-LPETG-1NOG in complex with antibodies, 5 μg of Sortase A-ligated GPCysRRLL-LPETG-1NOG trimer was incubated with 20 μg of 8.9F-scFv, 12.1F-scFv, 37.2D-scFv, 25.10C-Fab, 19.7E-Fab, or 37.7H-Fab at room temperature overnight. Excess scFv or Fab was removed using 100 KDa cut-off Amicon centrifugal filters (EMD Millipore). The concentrated complexes were further characterized by electron microscope analysis.

Eis-Hübinger A.M., Däumer M., Matz B, & Schneweis K.E. (1999). Evaluation of Three Glycoprotein G2-Based Enzyme Immunoassays for Detection of Antibodies to Herpes Simplex Virus Type 2 in Human Sera. Journal of Clinical Microbiology, 37(5), 1242-1246.

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Purification and Characterization of ToxRSp-ox

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ToxRp‐ox and ToxSp were purified according to the protocol described before and pooled in a 1:2 ratio with excess of ToxSp. To get rid of unbound ToxSp, the sample was further purified using a Superdex 200 Increase 10/300 column (GE healthcare). The resulting fractions were pooled and concentrated to 0.1 ml, containing 18 mg/ml of ToxRSp‐ox heterodimer using Amicon Ultra Centricons with a 3 kDa cutoff. The sample was loaded on a Superdex 200 Increase 10/300 column (GE healthcare) with a flow rate of 0.5 ml/min performed on a ÄKTA pure 25 (GE healthcare) connected to the miniDAWN Treos II MALS detector (Wyatt). The buffer used for the MALS‐SEC measurement contained 20 mM Tris, 150 mM sodium chloride, pH 7.5. The chromatogram resulted in a single peak eluting after 16.19 ml. The hydrodynamic radius determined by the MALS detector leads to a molecular weight of about 28 kDa, suggesting a heterodimer formation of ToxRSp‐ox.

Gubensäk N., Wagner G.E., Schrank E., Falsone F.S., Berger T.M., Pavkov‐Keller T., Reidl J, & Zangger K. (2021). The periplasmic domains of Vibriocholerae ToxR and ToxS are forming a strong heterodimeric complex independent on the redox state of ToxR cysteines. Molecular Microbiology, 115(6), 1277-1291.

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Characterizing PapA Polymerization by SEC

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Samples of purified PapD_His:PapA wild-type, Val18AzF, Asn96AzF, Asp126AzF, and Val155 AzF were left to polymerize at 20°C for 16 hr at a concentration of 38 μM. These samples (100 μl) were loaded consecutively onto a Superdex 200 10/300 increase column (GE Healthcare) to assess whether PapA has undergone DSE. To unambiguously identify the species responsible for each peak, a sample of PapD:PapA Val18AzF was run on a SEC-MALS instrument (Wyeth) also using a Superdex 200 10/300 increase column (GE Healthcare) and the molecular weight of each peak was derived using the manufacturer’s software.

Hospenthal M.K., Redzej A., Dodson K., Ukleja M., Frenz B., Rodrigues C., Hultgren S.J., DiMaio F., Egelman E.H, & Waksman G. (2016). Structure of a Chaperone-Usher Pilus Reveals the Molecular Basis of Rod Uncoiling. Cell, 164(1-2), 269-278.

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Characterization of Monoclonal Antibodies

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Twenty monoclonal antibodies discovered by single B cell technology were selected for characterization based on their binding specifies to HKU1-S, MERS-S and SARS-1-S (monospecific, bi-specific or tri-specific), diversity of heavy and light chain CDR3s, and high frequency rates in the B cell repertoire (independently isolated by the probes more than 2 times). The individual VH sequence of selected IgGs was cloned into a mammalian expression plasmid pVRC8400 containing HRV 3C cleavage site in the hinge, and human IgG1 Fc domain. VL sequences were also cloned into pVRC8400 with human CL. Paired VH and VL in a 1:1 ratio were co-transfected transiently into FreeStyle293F cells as previously described. The supernatant was harvested six days post-transfection and IgGs were purified with Protein A agarose (ThermoFisher). IgGs were eluted with 100 mM glycine, pH 3 into 1/10th volume 1 M Tris-HCl pH 8.0. IgGs were then buffer exchanged into PBS pH 7.4. Fabs were generated by digesting the IgGs with HRV 3C protease at 4°C. Fc was removed by passing digests over fresh Protein A agarose, leaving the Fab in the flowthrough, which was further purified by SEC using a Superdex 200 increase 10/300 column (GE Healthcare) in PBS buffer, pH 7.4.

Hsieh C.L., Werner A.P., Leist S.R., Stevens L.J., Falconer E., Goldsmith J.A., Chou C.W., Abiona O.M., West A., Westendorf K., Muthuraman K., Fritch E.J., Dinnon KH I.I.I., Schäfer A., Denison M.R., Chappell J.D., Baric R.S., Graham B.S., Corbett K.S, & McLellan J.S. (2021). Stabilized coronavirus spike stem elicits a broadly protective antibody. Cell Reports, 37(5), 109929.

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Superdex 200 FPLC Purification

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FPLC was performed as described in our previous report [29 ]. Briefly, an ÄKTA pure FPLC System (GE Healthcare, Munich, Germany) equipped with a Superdex 200 Increase 10/300 column (GE Healthcare, Munich, Germany) was used with PBS as mobile phase. After loading, HDL samples were separated with a constant flow of 0.5 mL/min, and 0.5 mL fractionation between of 9 to 13.5 mL elution volume were collected. The software UNICORN (GE Healthcare, Munich, Germany) was used for analysis.

Schilcher I., Ledinski G., Radulović S., Hallström S., Eichmann T., Madl T., Zhang F., Leitinger G., Kolb-Lenz D., Darnhofer B., Birner-Gruenberger R., Wadsack C., Kratky D., Marsche G., Frank S, & Cvirn G. (2019). Endothelial lipase increases antioxidative capacity of high-density lipoprotein. Biochimica et biophysica acta. Molecular and cell biology of lipids, 1864(10), 1363-1374.

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Site-Specific Protein Conjugation via Sialidase

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T-FcX–HIPS–azide (63.3 nmol, 1 equiv., 11.9 ml) and ST sialidase (316.9 nmol, 5 equiv., 16 ml), both in PBS buffer, were mixed together and concentrated to ~25 mg ml⁻¹ using 10,000 molecular weight cut-off Amicon spin filters. The final mixture (~0.92 ml) was incubated at 25 °C in the dark with shaking at 500 r.p.m. for 3 d. The reaction was monitored by SDS–PAGE and purified by size exclusion chromatography using a Superdex 200 increase 10/300 column on the ÄKTA pure chromatography system in PBS (GE Healthcare Life Sciences), followed by protein A chromatography. Final conjugates were buffer exchanged to PBS buffer with PD-10 columns and 0.2-μm was syringe filtered.

Gray M.A., Stanczak M.A., Mantuano N.R., Xiao H., Pijnenborg J.F., Malaker S.A., Miller C.L., Weidenbacher P.A., Tanzo J.T., Ahn G., Woods E.C., Läubli H, & Bertozzi C.R. (2020). Targeted glycan degradation potentiates the anticancer immune response in vivo. Nature chemical biology, 16(12), 1376-1384.

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Protein Molecular Weight Determination

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5 mg/ml (0.24 mM) of purified protein (wild-type Orn_Vc, Orn_Vc-D¹²A, or Orn_Vc-R¹³⁰A) was injected onto a Superdex 200 Increase 10/300 column (GE Healthcare) equilibrated with gel filtration buffer (25 mM Tris-Cl, pH 7.5, 150 mM NaCl). Samples were run continuously at a flow rate of 0.75 ml/min through the gel filtration column coupled to a static 18-angle light scattering detector (DAWN HELIOS-II) and a refractive index detector (Optilab T-rEX), with data being collected every second. Data analysis was performed with ASTRA VI, yielding the molar mass and mass distribution (polydispersity) of the sample, using monomeric BSA (Sigma; 5 mg/ml) to normalize the light scattering detectors and as a control sample.

Kim S.K., Lormand J.D., Weiss C.A., Eger K.A., Turdiev H., Turdiev A., Winkler W.C., Sondermann H, & Lee V.T. (2019). A dedicated diribonuclease resolves a key bottleneck for the terminal step of RNA degradation. eLife, 8, e46313.

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SEC-MALLS Analysis of Timeless Proteins

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For SEC-MALLS experiments a Superdex 200 Increase 10/300 column (GE Healthcare) was coupled with a DAWN HELEOS II MALLS detector with a 664 nm laser light source, eight fixed-angle detectors and an Optilab T-rEX differential refractometer (Wyatt Technology) at 25°C. A total of 1–2 mg/ml (100 μl injection) of Timeless(1–1208_Δ239-330) and Timeless(1–463_Δ239-330) were analysed in 25 mM Hepes 7.2, 150 mM KCl, 50 mM Arginine, 50 mM Glutamate. The collected data were analysed and processed using ASTRA 6 (Wyatt Technology).

Holzer S., Degliesposti G., Kilkenny M.L., Maslen S.L., Matak-Vinkovíc D., Skehel M, & Pellegrini L. (2017). Crystal structure of the N-terminal domain of human Timeless and its interaction with Tipin. Nucleic Acids Research, 45(9), 5555-5563.

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SEC-MALS Analysis of Purified Atg40

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SEC-MALS was performed with the HPLC system (Shimadzu) equipped with a Shimadzu LC-20AD pump, a UV detector (SDP-20A), a MALS detector (DAWN HELEOS II, Wyatt Technology), and a differential refractive index (RI) detector (RI-501, Shodex). The sample was applied to Superdex 200 increase 10/300 column (GE Healthcare) equilibrated in buffer J at room temperature. The data analysis was carried out using Wyatt ASTRA software using a dn/dc value (protein 0.1853, DDM 0.1435) and UV Ext. coefficient (purified Atg40 1.220).

Mochida K., Yamasaki A., Matoba K., Kirisako H., Noda N.N, & Nakatogawa H. (2020). Super-assembly of ER-phagy receptor Atg40 induces local ER remodeling at contacts with forming autophagosomal membranes. Nature Communications, 11, 3306.

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