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Superdex 200 10 300 column

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The Superdex 200 10/300 column is a size exclusion chromatography column used for the separation and purification of macromolecules such as proteins, peptides, and other biomolecules. It has a matrix of cross-linked agarose and dextran, and a bed volume of 24 mL. The column is designed for use with FPLC or HPLC systems and is suitable for a wide range of molecular weight analytes.

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Lab products found in correlation

Astra 6, by Wyatt Technology (8 mentions) Astra software, by Wyatt Technology (7 mentions) Akta protein purification system, by GE Healthcare (6 mentions) Optilab t rex, by Wyatt Technology (6 mentions) Optilab t rex refractive index detector, by Wyatt Technology (3 mentions) Protein a affinity chromatography, by GE Healthcare (3 mentions) Expi293 expression system, by Thermo Fisher Scientific (3 mentions) Pierce fab preparation kit, by Thermo Fisher Scientific (3 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions) Minidawn tristar, by Wyatt Technology (2 mentions) Ultra concentrators, by GE Healthcare (2 mentions) Kta purifier, by GE Healthcare (2 mentions) 0.22 μm filter, by Merck Group (2 mentions) Protease inhibitor cocktail, by Roche (2 mentions) Hispur cobalt resin, by Thermo Fisher Scientific (2 mentions) Hplc 1260 infinity lc, by Agilent Technologies (2 mentions) Minidawn treos multiangle static light scattering device, by Wyatt Technology (2 mentions) Astra6 program, by Wyatt Technology (2 mentions) Superose 6 10 300 column, by GE Healthcare (2 mentions) Ni2 nta, by GE Healthcare (2 mentions)

198 protocols using superdex 200 10 300 column

1

Purification and Characterization of CA09-15 mAb

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The monoclonal antibody CA09-15 in mouse ascites was obtained through BEI Resources (Item# NR-28668). For purification, the sample was diluted in excess Protein A IgG Binding Buffer (Thermo Scientific) and added to a gravity flow column containing 0.5 mL of packed Protein A resin equilibrated with Binding Buffer. The column was washed with 3 mL of Binding Buffer, then the protein eluted with 3 mL of Protein A IgG Elution Buffer (Thermo Scientific) divided into three fractions. To neutralize the pH, 100 μL of 1 M Tris pH 8.5 was added to each 1 mL fraction. The presence of antibody was confirmed by SDS-PAGE, then the first two fractions combined and concentrated. CA09-15 mAb for BLI studies was further purified by size exclusion chromatography on a Superdex 200 10/300 column (GE Healthcare) equilibrated in PBS and quantified using an assumed extinction coefficient of 1.4 g/L and a 150 kD molecular weight.
Fab fragments were generated and purified using the Pierce™ Fab Preparation Kit according to the manufacturer’s instructions (Thermo Scientific) and the resulting product was further polished by size exclusion chromatography on a Superdex 200 10/300 column (GE Healthcare) equilibrated in PBS. The protein was quantified using an assumed extinction coefficient of 1.4 g/L and a 50 kD molecular weight.
Dzimianski J.V., Han J., Sautto G.A., O’Rourke S.M., Cruz J.M., Pierce S.R., Ecker J.W., Carlock M.A., Nagashima K.A., Mousa J.J., Ross T.M., Ward A.B, & DuBois R.M. (2023). Structural insights into the broad protection against H1 influenza viruses by a computationally optimized hemagglutinin vaccine. Communications Biology, 6, 454.
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2

Purification of Neurochondrin Protein

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Purified neurochondrin (200 μL) was separated on a Superdex™ 200 10/300 column (GE Healthcare, Freiburg, Germany) in buffer A at a flow rate of 0.5 mL/min. The Superdex™ 200 10/300 column was calibrated using the gel filtration calibration kit (GE Healthcare, Freiburg, Germany) according to the manufacturer’s protocol.
Hermann J.S., Skroblin P., Bertinetti D., Hanold L.E., von der Heide E.K., Wagener E.M., Zenn H.M., Klussmann E., Kennedy E.J, & Herberg F.W. (2015). Neurochondrin is an atypical RIIα-specific A-kinase anchoring protein. Biochimica et biophysica acta, 1854(10 0 0), 1667-1675.
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3

FANCD2-FANCI Complex Formation Analysis

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A volume of 300 μl of purified D2–I, FANCD2 and FANCI at 1
μM were sequentially run on a Superdex 200 10/300 column (GE Healthcare
Life Sciences) equilibrated in 50 mM HEPES pH 7.5, 150 mM NaCl and 1 mM TCEP. To
investigate the exchange of FANCD2 and FANCI, we mixed and incubated 0.5
μM FANCD2 with 1 μM FANCI for 30 min at room temperature in a
total volume of 300 μl. The mix was then run on a Superdex 200 10/300
column (GE Healthcare Life Sciences) equilibrated in 50 mM HEPES pH 7.5, 150 mM
NaCl and 1 mM TCEP. Fractions were analyzed by SDS-PAGE using 4–12%
NuPAGE Bis-Tris gels (ThermoFisher Scientific).
Alcón P., Shakeel S., Chen Z.A., Rappsilber J., Patel K.J, & Passmore L.A. (2020). FANCD2–FANCI is a clamp stabilized on DNA by monoubiquitination of FANCD2 during DNA repair. Nature structural & molecular biology, 27(3), 240-248.
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4

FANCD2-FANCI Complex Formation Analysis

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A volume of 300 μl of purified D2–I, FANCD2 and FANCI at 1
μM were sequentially run on a Superdex 200 10/300 column (GE Healthcare
Life Sciences) equilibrated in 50 mM HEPES pH 7.5, 150 mM NaCl and 1 mM TCEP. To
investigate the exchange of FANCD2 and FANCI, we mixed and incubated 0.5
μM FANCD2 with 1 μM FANCI for 30 min at room temperature in a
total volume of 300 μl. The mix was then run on a Superdex 200 10/300
column (GE Healthcare Life Sciences) equilibrated in 50 mM HEPES pH 7.5, 150 mM
NaCl and 1 mM TCEP. Fractions were analyzed by SDS-PAGE using 4–12%
NuPAGE Bis-Tris gels (ThermoFisher Scientific).
Alcón P., Shakeel S., Chen Z.A., Rappsilber J., Patel K.J, & Passmore L.A. (2020). FANCD2–FANCI is a clamp stabilized on DNA by monoubiquitination of FANCD2 during DNA repair. Nature structural & molecular biology, 27(3), 240-248.
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5

Size Exclusion Chromatography of His6-GlnR

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Purified His6-GlnR from E. coli was used for size-exclusion chromatography. Analytical size-exclusion chromatography was carried out on an Akta purifier system equipped with a Superdex 200 column 10/300 (geometric column volume of 24 mL GE Healthcare). The running buffer contains 50 mM Tris-HCl (pH 7.4) and 300 mM NaCl. His6-tagged GlnR was diluted on running buffer to reach a concentration of 2 mg/ml. 1 mL filtered and centrifuged sample was injected at a flow rate of 0.3 ml/min. Purified His6-GlnR from E. coli was used for size-exclusion chromatography. Analytical size-exclusion chromatography was carried out on an Akta purifier system equipped with a Superdex 200 column 10/300 (geometric column volume of 24 mL GE Healthcare). The running buffer contains 50 mM Tris-HCl (pH 7.4) and 300 mM NaCl. His6-GlnR was diluted on running buffer to reach a concentration of 2 mg/ml. 1 mL filtered and centrifuged sample was injected at a flow rate of 0.3 ml/min. The apparent molecular weights of proteins were estimated after calibration of the column with standard proteins: thyroglobulin (670 kDa), globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa), vitamin B12 (1.35 kDa) (Bio-Rad gel filtration standard).
Wang T., Zhao X., Shi H., Sun L., Li Y., Li Q., Zhang H., Chen S, & Li J. (2018). Positive and negative regulation of transferred nif genes mediated by indigenous GlnR in Gram-positive Paenibacillus polymyxa. PLoS Genetics, 14(9), e1007629.
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6

Production and Purification of FZD and LRP Proteins

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Fc-tagged fusions of FZD1 (5988-FZ-050), FZD2 (1307-FZ-050), FZD4 (5847-FZ-050), FZD5 (1617-FZ-050), FZD7 (6178-FZ-050), FZD8 (6129-FZ-050), FZD9 (9175-FZ-050), FZD10 (3459-FZ-050) were purchased from R and D Systems. The Fc-tagged ECD of FZD6 (residues 19–132, Uniprot O60353-1) was expressed and purified from Expi293 cells using the pFUSE-hIgG1-Fc2 vector (invivogen) and the single protomer species was separated from aggregated protein by size exclusion chromatography on a Superdex 200 (10/300) column (GE Healthcare). Fc-tagged ECD fusion proteins of human (1505-LR-025) and mouse (2960-LR-025) LRP6 and mouse LRP5 (7344-LR-025/CF) were purchased from R and D Systems. WNT1 (SRP4754-10ug), WNT2b (3900-WN-010/CF), WNT5a (645-WN-010/CF) and WNT3A (5036-WN-010/CF) were purchased from R and D Systems, and WNT3A conditioned media was prepared as described (Lui et al., 2011 (link)). Other proteins and chemicals were purchased from the following suppliers: FcRN (R and D, 8693-FC), C1q (Sigma, C1740), CD16a (R and D, 4325-FC), CD32a (R and D, 1330 CD/CF), CD64 (R and D, 1257-FC), LGK974 (Cayman Chemicals), C59 (Dalriada Therapeutics), and CHIR99021 (Sigma Aldrich).
Tao Y., Mis M., Blazer L., Ustav M J.n.r., Steinhart Z., Chidiac R., Kubarakos E., O'Brien S., Wang X., Jarvik N., Patel N., Adams J., Moffat J., Angers S, & Sidhu S.S. (2019). Tailored tetravalent antibodies potently and specifically activate Wnt/Frizzled pathways in cells, organoids and mice. eLife, 8, e46134.
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7

Structural Analysis of VDR-RXR-DNA Complex

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Synchrotron X-ray data were collected on a Pilatus 1M detector at the ESRF beamline BM29 (51 ). 100 μl of VDR–RXRΔNTD-DNA, MED1 (50–660) and their complex at concentrations 8.5–10 mg.mL−1 in 25 mM HEPES pH 7.5, 150 mM NaCl, 5% glycerol, 2 mM MgCl2, 2 mM TCEP were loaded onto a GE Healthcare Superdex 200 10/300 column (equilibrated in the same buffer) at a flow rate of 1 mL.min−1. A scattering profile was integrated every second. Frames were selected based on the examination of the SEC profile together with the calculated Rg and Dmax values. The SAXS data were averaged and processed by standard procedures using PRIMUS (52 ). The forward scattering I(0) and the radii of gyration Rg were evaluated using the Guinier approximation assuming that at very small angles (s < 1.3/Rg) the intensity is represented as I(s) = I(0) exp(–(sRg)2/3). These parameters were also computed from the entire scattering pattern using the indirect transform package GNOM (53 ) which also provides the maximum dimension of the particle Dmax and the distance distribution function p(r). The program SASREF (54 (link)) was employed for molecular rigid body modeling of the VDR–RXR–DNA complex, based on SAXS and cryoEM structures (48 (link),55 (link)). The final fits of the model scattering to the experimental data were computed using CRYSOL (56 ).
Belorusova A.Y., Bourguet M., Hessmann S., Chalhoub S., Kieffer B., Cianférani S, & Rochel N. (2020). Molecular determinants of MED1 interaction with the DNA bound VDR–RXR heterodimer. Nucleic Acids Research, 48(19), 11199-11213.
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8

Gel Filtration Fractionation and Analysis

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IEX fractions of highest DC-suppressive activity were pooled for Gel filtration chromatographic fractionation. The gel filtration was performed on an Äkta Explorer machine (GE Healthcare) using a Superdex 200 10/300 column (GE Healthcare) equilibrated with 2 column volumes PBS (pH 7). Approximately 500 μl of 2.5 mg/ml protein solution was applied. Loading and elution was done in the same buffer using a flow rate of 0.4 ml/min. Protein elution was monitored by measuring the absorption at 280 nm and 400 μl fractions were collected. The eluted fractions were probed by Bicinchoninic acid assay for protein quantification before testing in the in vitro DC stimulation assay. The tested fractions were analyzed by SDS-PAGE. Immunosuppressive fractions (3) and non-active fractions (4) were lyophilized and the protein composition analyzed by LC-MS/MS.
Vendelova E., Camargo de Lima J., Lorenzatto K.R., Monteiro K.M., Mueller T., Veepaschit J., Grimm C., Brehm K., Hrčková G., Lutz M.B., Ferreira H.B, & Nono J.K. (2016). Proteomic Analysis of Excretory-Secretory Products of Mesocestoides corti Metacestodes Reveals Potential Suppressors of Dendritic Cell Functions. PLoS Neglected Tropical Diseases, 10(10), e0005061.
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9

Anionic Phospholipid Incorporation in Nanodiscs

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Nanodiscs containing PYR-PC were prepared as described for the POPC-PYR nanodiscs, albeit with the addition of differing ratios of anionic phospholipids (POPS). Nanodiscs containing 30% and 60% POPS were prepared by mixing POPC/PYR/POPS in a molar ratio of 68.5:1.5:30 and 38.5:1.5:60, respectively. Each POPC/PYR/POPS mixture was added to MSP1D1 in a 65:1 ratio and allowed to equilibrate for 1 hour at 4°C before detergent removal with Amberlite Biobeads. The assembled nanodiscs were isolated using SEC-HPLC on a Superdex 200 10/300 column (GE Life Sciences, Piscataway, NJ) with a mobile phase consisting of 100 mM phosphate buffer (pH 7.4) and a 0.5 ml/min flow rate
McDougle D.R., Baylon J.L., Meling D.D., Kambalyal A., Grinkova Y.V., Hammernik J., Tajkhorshid E, & Das A. (2015). Incorporation of Charged Residues in the CYP2J2 F-G Loop Disrupts CYP2J2-Lipid Bilayer Interactions. Biochimica et biophysica acta, 1848(10 0 0), 2460-2470.
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10

Reconstitution of POPC Nanodiscs

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POPC was solubilized with cholate (50 mM) and mixed with the membrane scaffold protein ΔTrp-MSP1D1 (devoid of Trp ε280 = 10,040 mM−1 cm−1) in a 65:1 ratio before rocking the mixture at 4°C for one hour. Nanodisc assembly was initiated upon removal of detergents using of Amberlite Biobeads (Supelco). The homogenous nanodisc assembly was isolated using SEC-HPLC with a Superdex 200 10/300 column (GE Life Sciences, Piscataway, NJ) and a mobile phase consisting of 100 mM phosphate buffer (pH 7.4) and a 0.5 ml/min flow rate. The isolated nanodiscs were then concentrated with Amicon Ultra (10,000 MWCO) centrifugal filters (Millipore) to a final concentration of 500 μM.
McDougle D.R., Baylon J.L., Meling D.D., Kambalyal A., Grinkova Y.V., Hammernik J., Tajkhorshid E, & Das A. (2015). Incorporation of Charged Residues in the CYP2J2 F-G Loop Disrupts CYP2J2-Lipid Bilayer Interactions. Biochimica et biophysica acta, 1848(10 0 0), 2460-2470.
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