The fractions obtained from SEC were pooled and concentrated to ∼0.5 mg/ml. SEC–MALS was performed using 20 mM Tris pH 7.5 and 150 mM NaCl buffer–equilibrated analytical Superdex 200 Increase 10/300 GL gel filtration column (GE Healthcare) on a Shimadzu HPLC. Protein peaks resolved after size exclusion were subjected to in-line refractive index (Waters Corp) and MALS (mini DAWN TREOS, Wyatt Technology Corp) detection to estimate the molar mass. The data acquired from UV, MALS, and refractive index were analyzed using ASTRA 6.1 software (https://www.wyatt.com/products/software/astra.html ; Wyatt Technology).
Superdex 200 increase 10 300 gl gel filtration column
Manufactured by GE Healthcare
Sourced in United States
The Superdex 200 Increase 10/300 GL gel filtration column is a laboratory equipment product designed for size-exclusion chromatography. It is used for the separation and purification of biomolecules, such as proteins and peptides, based on their molecular size and shape. The column has a bed volume of 24 ml and is compatible with standard FPLC and HPLC systems.
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Lab products found in correlation
Akta avant system, by GE Healthcare (3 mentions)
Akta avant, by Cytiva (3 mentions)
Astra 6, by Wyatt Technology (2 mentions)
Minidawn treos, by Wyatt Technology (1 mentions)
270 dual detector, by Malvern Panalytical (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Amicon ultra, by Merck Group (1 mentions)
Amylose agarose resin, by New England Biolabs (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Dawn heleos 2 mals instrument, by Wyatt Technology (1 mentions)
Kta fast protein liquid chromatography system, by GE Healthcare (1 mentions)
Optilab t rex differential refractometer, by Wyatt Technology (1 mentions)
N ethylamine, by Merck Group (1 mentions)
Akta fplc system, by GE Healthcare (1 mentions)
Ni nta superflow column, by Qiagen (1 mentions)
6550 ifunnel q tof lc ms, by Agilent Technologies (1 mentions)
Zorbax extend c18 column, by Agilent Technologies (1 mentions)
Poly prep column, by Bio-Rad (1 mentions)
Vitrobot mark 4, by Thermo Fisher Scientific (1 mentions)
Falcon 3 direct electron detector, by Thermo Fisher Scientific (1 mentions)
16 protocols using superdex 200 increase 10 300 gl gel filtration column
1
Protein Characterization by SEC-MALS
2
Size Exclusion Chromatography of Anti-TF mAb-7t15-21s Complex
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Example 72
To determine if anti-tissue factor monoclonal antibody and 7t15-21s can form an antibody-fusion-molecule complex, analytical size exclusion chromatography (SEC) was performed. A Superdex 200 Increase 10/300 GL gel filtration column (from GE Healthcare) was connected to an AKTA Avant system (from GE Healthcare). The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.7 mL/min. Samples of the anti-TF mAb (1 mg/mL), 7t15-21s (1 mg/mL), and a mixture of combined at a 1:1 ratio, so the final concentration of each protein is 0.5 mg/mL) were in PBS. Each sample was injected into the Superdex 200 column using a capillary loop, and analyzed by SEC. The SEC chromatograph of each sample was shown in
3
Characterizing Antibody-Antigen Complex Formation
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Example 45
To determine if anti-tissue factor monoclonal antibody and 7t15-21s can form an antibody-fusion-molecule complex, analytical size exclusion chromatography (SEC) was performed. A Superdex 200 Increase 10/300 GL gel filtration column (from GE Healthcare) was connected to an AKTA Avant system (from GE Healthcare). The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.7 mL/min. Samples of the anti-TF mAb (1 mg/mL), 7t15-21s (1 mg/mL), and a mixture of combined at a 1:1 ratio, so the final concentration of each protein is 0.5 mg/mL) were in PBS. Each sample was injected into the Superdex 200 column using a capillary loop, and analyzed by SEC. The SEC chromatograph of each sample was shown in
4
Antibody-Fusion Protein Complex Analysis
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Example 21
To determine if anti-tissue factor monoclonal antibody and 7t15-21s can form an antibody-fusion-molecule complex, analytical size exclusion chromatography (SEC) was performed. A Superdex 200 Increase 10/300 GL gel filtration column (from GE Healthcare) was connected to an AKTA Avant system (from GE Healthcare). The column was equilibrated with 2 column volumes of PBS. The flow rate was 0.7 mL/min. Samples of the anti-TF mAb (1 mg/mL), 7t15-21s (1 mg/mL), and a mixture of combined at a 1:1 ratio, so the final concentration of each protein is 0.5 mg/mL) were in PBS. Each sample was injected into the Superdex 200 column using a capillary loop, and analyzed by SEC. The SEC chromatograph of each sample was shown in
5
SEC-MALS Analysis of Holo and Apo HphA
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SEC-MALS analysis on holo and apo HphA was done using a Malvern Viscotek GPCmax system connected to a Superdex 200 increase 10/300 GL gel filtration column (GE Healthcare). The column was equilibrated with buffer containing 20 mM Hepes pH 7.5, 100 mM NaCl. The scattered light intensity of the column eluate was recorded using a VE 3580 RI and Malvern 270 Dual detector. Bovine serum albumin at a concentration of 1 mg/mL was used for detector normalization. Molecular weights were calculated from Zimm plots using a protein refractive index increment (dn/dc) of 0.185 mL/g using the OmniSEC 5.10 software (Malvern).
6
Purification of Mouse Naa10 and Naa12
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All constructs were expressed in Rosetta (DE3)pLysS competent Escherichia coli cells. Cells were grown in LB-media to OD600 0.6–0.7 prior to inducing protein expression with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 18°C for ~16 hr. All subsequent purification steps were carried out at 4°C. Cells were isolated by centrifugation and lysed in lysis buffer containing 25 mM Tris, pH 8.0, 150 mM NaCl, 10 mM β-mercaptoethanol (β-ME), 10 µg/mL phenylmethanesulfonylfluoride (PMSF), and DNase. The lysate was clarified by centrifugation and incubated with amylose agarose resin (New England Biolabs) for 1 hr before washing the resin with ≥100 column volumes of lysis buffer and then eluted with 10-column volumes of lysis buffer supplemented with 20 mM maltose. The resulting eluent was pooled and concentrated to ~10 mg/mL (30 kDa concentrator; Amicon Ultra, Millipore) such that 500 µL was loaded onto a Superdex 200 Increase 10/300 GL gel filtration column (GE Healthcare). The gel filtration run was performed in sizing buffer containing 25 mM HEPES, pH 7.0, 200 mM NaCl, and 1 mM TCEP. After confirming the purity of the peak fractions at ~14 mL by denaturing SDS-PAGE (15% acrylamide), peak fractions were concentrated to 0.6 (6.1 µM) WT mouse Naa10 and 0.3 mg/mL (3.5 µM) WT mouse Naa12, as measured by UV280 (Nanodrop 2000; Thermo Fisher Scientific), and stored at 4°C.
7
Purification of Fluorescent Proteins
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Colonies of E. coli strain JM109(DE3) carrying an FP plasmid were transferred to 50 mL 2×YT medium and cultured at 24 °C for 2 d with vigorous shaking. Cells were collected by centrifugation, suspended in buffer A [100 mM sodium phosphate (pH 8.0) and 200 mM sodium chloride] containing a protease inhibitor cocktail (Roche), and then disrupted by sonication. Cell-free extracts, obtained by centrifugation at 12,000 × g for 60 min, were applied to a Talon Sepharose columns (Clontech) pre-equilibrated with buffer A. After washing the column with buffer A containing 5 mM imidazole, the his-tagged proteins were eluted by increasing the imidazole concentration to 150 mM. Fractions containing FP were concentrated, followed by application to a Superdex 200 Increase 10/300 GL gel-filtration column (GE Healthcare) pre-equilibrated with buffer B [20 mM Tris-HCl (pH 8.0) and 150 mM sodium chloride]. Fractions containing FP were collected and stored at 4 °C.
8
Oligomeric State Determination of MINERVA Proteins
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The oligomeric states of the recombinant MINERVAFL and MINERVAΔC proteins were assessed by SEC–MALS experiments using an ÄKTA fast protein liquid chromatography (FPLC) system (GE Healthcare, Chicago, IL, USA) connected to a Wyatt DAWN Heleos II MALS instrument and a Wyatt Optilab T-rEX differential refractometer. A Superdex 200 Increase 10/300 GL gel-filtration column (GE Healthcare, Chicago, IL, USA), which was pre-equilibrated with buffer A containing 20 mM Tris-HCl (pH 8.0) and 200 mM NaCl, was normalized using ovalbumin. The proteins (2 mg) were injected at a flow rate of 0.5 mL min−1. Data were analyzed using the Zimm model for fitting static light-scattering data and were graphed using Easy Analytic Software Inc. (EASI) graph with a UV peak in the ASTRA 6 software (Wyatt, Santa Barbara, CA, USA).
9
Antibody Fragment Preparation and Purification
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Digestion of human IgG4 mAbs was performed using immobilized FabRICATOR (A0-FR6-100, Genovis) in 10 mM PBS pH 7.4 at 37°C for 1 h and antibody fragments were separated using spin columns. Digested F(ab′)2 was purified using Fc based capture select spin columns. Purified F(ab′)2 fragments were reduced to Fab′ fragments using the mild reducing agent 2-merceptoethylamine hydrochloride (M6500-25G, Sigma) at 37°C for 90 min and adjusted to a final concentration of 50 mM. Reduced Fab′ fragments were then buffer exchanged using a G25 Sephadex® in PD10 desalting column (17085101, GE Healthcare) followed by alkylation of reduced SH groups using N-ethylamine (E3876-25G, Sigma). Alkylated Fab′ fragments were gel filtered using a Superdex 200 Increase 10/300 GL gel filtration column (28-9909-44, GE Healthcare) to remove impurities, with 99% monomeric purity measured by analytical size exclusion chromatography.
10
Purification and Size Exclusion Chromatography of atACS
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Purified atACS proteins were subjected to size exclusion gel filtration chromatography using an AKTA FPLC system (GE Healthcare Life Sciences, Pittsburg, PA) with a Superdex 200 Increase 10/300 GL gel filtration column (GE Healthcare Life Sciences, Pittsburg, PA). A 100 µL aliquot of purified atACS protein (4–10 mg/mL) was injected into the prepacked column, and chromatography was conducted with a buffer consisting of 10 mM HEPES-KOH, pH 7.5, 10 mM KCl, 2 mM TCEP, 10% glycerol, eluted at a rate of 0.4 mL/min; the eluate was monitored using a UV absorbance detector, at 280 nm.
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