Size-exclusion HPLC was carried out on a Superose 12 column (10 Â 300 mm, GE Healthcare, Buckinghamshire, England) as previously described. 14 Briefly, 200 mL of serum were injected. The elution buffer was 150 mM NaCl and 16 mM phosphate buffer (pH 7.4). The flow rate was 0.5 mL/min and the fraction volume was 0.8 mL. The Superose 12 column was calibrated using a Gel Filtration Calibration Kit with low-molecular-weight and highmolecular-weight proteins (GE Healthcare). Protein and CA19-9 concentrations were monitored at 280 nm using the Akta Explorer System (GE Healthcare) and an AIA 1800 analyser, respectively.
Superose 12 column
Manufactured by GE Healthcare
Sourced in Sweden, United Kingdom, United States
The Superose 12 column is a gel filtration chromatography column used for the separation and purification of proteins, peptides, and other biomolecules. It is designed to provide efficient and reproducible separations based on molecular size. The column features a stable and inert matrix material that allows for a wide range of sample applications and operating conditions.
Automatically generated - may contain errors
Lab products found in correlation
Size exclusion standard, by Bio-Rad (2 mentions)
Waters 2487, by Waters Corporation (1 mentions)
L 1 tosylamido 2 phenylethyl chloromethyl ketone treated trypsin, by Merck Group (1 mentions)
Smart system, by GE Healthcare (1 mentions)
Atpγs, by Merck Group (1 mentions)
Kta purifier fplc, by GE Healthcare (1 mentions)
Pd 10 desalting column, by GE Healthcare (1 mentions)
Amicon ultra centrifugal filter, by Merck Group (1 mentions)
Mini protean tgx, by Bio-Rad (1 mentions)
Tdamax, by Malvern Panalytical (1 mentions)
Biologic duo flow protein purification system, by Bio-Rad (1 mentions)
Limulus amebocyte lysate kit qcl 1000, by Lonza (1 mentions)
Detoxi gel endotoxin removing gel, by Thermo Fisher Scientific (1 mentions)
Microcon centrifugal filter, by Merck Group (1 mentions)
Akta fast protein liquid chromatography system, by GE Healthcare (1 mentions)
Glutathione agarose beads, by Merck Group (1 mentions)
Prescission protease, by GE Healthcare (1 mentions)
Protein g sepharose, by Cytiva (1 mentions)
Protein g sepharose, by Merck Group (1 mentions)
Kta pure chromatography system, by GE Healthcare (1 mentions)
40 protocols using superose 12 column
1
Serum Protein and CA19-9 Analysis
2
Size-exclusion Chromatography of Cancer Sera
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Size-exclusion chromatography was performed as described previously [32 (link),33 (link)]. In brief, 200 μL of sera from MDS, breast, prostate cancer patients and blood donors was diluted to 1:1 in a buffer containing 0.01 M Hepes, 0.15 M NH4Cl and 0.02% NaN3 prior to injection into a Superose 12 column (1.0 × 30 cm, GE Health care, Sweden). The column was equilibrated with the same buffer and eluted at flow rate of 0.4 mL/min. Twenty four fractions (0.4 mL) were collected, and the TK1 protein content in the fractions determined and compared with standards of different molecular weights: α2-macroglobulin (720 kDa), β amylase (200 kDa), bovine serum albumin (66 kDa), ova albumin (45 kDa) and horse myosin (17 kDa).
3
Protein and Metabolite Fractionation Protocol
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A protein and a metabolite fraction was generated by separating an M. extorquens PA1 cell lysate (cultivated as described above, lysed using French press) using size exclusion chromatography with a Superose 12 column (GE Healthcare) and PBS (described above) as buffer. Standard solutions (blue dextran, ferritin, cytochrome c, aprotinin, and vitamin B12) were used to determine the fractions that correspond to molecules larger (protein fraction) and smaller (metabolite fraction) than ∼10 kDa. Fractions were pooled and heat-denatured (99 °C, 20 min) to release bound MYFR. The samples were spiked with a custom-synthesized peptide (by Thermo Fisher Scientific) consisting of 20 γ-linked l -glutamic acids connected with an l -tyrosine (γE20Y) as an internal standard. This peptide behaves chemically very similar to MYFR (5 (link)) and allowed to control for matrix effects. For desalting and enrichment of MYFR, the samples were diluted with water and concentrated ∼10-fold with a centrifugal filter with a molecular mass cutoff of 1 kDa (Microsep Advance with Omega membrane, Pall Life Sciences). This step was repeated four more times. The retentate was dried and measured by LC-MS. MYFR peak areas were normalized to the peak area of the internal standard γE20Y.
4
Gel-filtration Assay for Protein Size
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Gel-filtration assays were performed using the ÄKTAFPLC on a Superose 12 column (GE Healthcare) in 20 mmol/l Tris/HCl, pH 7.2, and 50 mmol/l NaCl. Proteins were added in a 500-µl super loop and separated by a flow rate of 500 µl/min. The eluate was collected in 500-µl fractions. Eluted proteins were detected at 280 nm (I9-12 construct) or 220 nm (PEVK construct), and the elution profiles (absorption versus elution volume) were recorded. For size determination, results were compared with those obtained with a reference protein of known size (66 kD BSA). Experiments were run at least three times for each construct with similar results (only one dataset shown).
5
Directed Evolution of Alcohol Dehydrogenase
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The mutation H103Y was introduced to the plasmid pPICZB_AmPDH by site-directed mutagenesis as described in section 2.3 using the overlapping primers H103Yfw and H103Yrev. Prior to transformation into electro-competent P. pastoris strain X33 the plasmid was linearized with PmeI at 37°C for at least 2 h and purified. Transformants were selected on YPD-Zeocin plates and the integration of the gene was verified by colony PCR. PDH variant H103Y was produced in a 7-L fermenter (MBR, Wetzikon, Switzerland) with an initial volume of 4 L basal salts fermentation medium and purified in a 3-step protocol as described before [6] (link). Fractions of the highest purity were pooled, concentrated and frozen in liquid nitrogen for storage at -30°C. Gel filtration pool 1 was used for all characterizations. Wild-type AmPDH protein was produced and purified as described previously [36] (link). In contrast to mutant H103Y, AmPDH is present in the reduced state after purification, oxidized enzyme was generated according to [13] (link) except using a Superose 12 column (GE Healthcare, Chalfont St. Giles, UK) and 50 mM potassium phosphate buffer pH 7.5 containing 150 mM NaCl.
6
Size Exclusion Chromatography of Leukemia Samples
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Size exclusion chromatography was performed as described elsewhere [38 (link),39 (link)] using a Superose 12 column (1.0 × 30 cm; GE Healthcare, Uppsala, Sweden) attached to fast protein liquid chromatography (FPLC) equipment (GE Healthcare, Uppsala, Sweden). Acute lymphocytic leukemia cell extract, ALL sera (100 μl), CMT tissue extract, CMT sera (from dog No. 23), and sera from a healthy dog (No. 5) (200 μl) were diluted in hydroxyethyl-piperazineethane-sulfonic acid (HEPES), pH 7.6, buffer (0.01 M) (NH4Cl, 0.15 M, and NaN3, 0.02%) and applied on the column. Standard proteins were α2-macroglobulin, 720 kDa; β-amylase, 200 kDa; bovine serum albumin (BSA), 66 kDa; ova albumin, 45 kDa; and horse myosin, 17 kDa.
7
Size Exclusion Chromatography of Polypeptides
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MWD reflects the general distribution of the polypeptides in a complex mixture according to their size and relative abundance. The retention time of each component of the mixture on the size exclusion chromatographic (SEC) column depends on its hydrodynamic size, rather than the primary structure of the constituents. The Mylan FOGA samples were diluted to 4 mg/mL and evaluated using a Superose 12 column (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) with pH 1.5 (acidic) phosphate buffer mobile phase at 0.5 mL/min, with UV detection at 208 nm, and then compared to Copaxone release specifications.
8
Analytical SEC-MALS for Protein Molar Mass
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Molar masses of 6HB and HR1 were analyzed by analytical SEC with inline MALS (Wyatt-925- H2HC, DAWN Heleos; Wyatt Technology Inc.), refractive index (Wyatt-215-TRXH; Wyatt Technology Inc.), and UV (Waters 2487; Waters Corporation) detectors. Samples were applied (125 μl) to a pre-equilibrated Superose-12 column (1.0 × 30 cm; GE Healthcare) and eluted at a flow rate of 0.5 ml/min at room temperature in 20 mM sodium phosphate at pH 6.0 and 30 mM NaCl. Molar masses were calculated using the Astra software provided with the instrument. Calculated masses of 6HB (65 μM injection) and HR1 (90 μM injection) are 29.4 and 19.6 kDa, respectively.
9
Hepatic Lipid Extraction and Analysis
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Extraction of hepatic lipids, and levels of plasma lipids, lipoproteins, and chemistries were measured as follows. The method of Folch et al38 (link) was used to extract hepatic lipids from liver (∼100 mg). Levels of hepatic TG, cholesterol, and free cholesterol were measured in liver using enzymatic assays (Infinity; Thermo Fisher Scientific, Waltham, MA) and normalized to sample weights. The hepatic lipids are expressed as mg/g liver tissue (wet weight). Blood was collected into tubes containing heparin from the tail veins of the rats. Plasma was isolated by centrifugation at 2000g for 10 minutes at 4°C. To determine the distribution of cholesterol and TG among circulating lipoproteins, plasma from 4 rats was pooled (total volume, 300 μL) and fractionated using fast performance liquid chromatography on a Superose 12 column (GE Healthcare). Cholesterol and TG levels in each fraction were measured enzymatically as described earlier. Aspartate aminotransferase and alanine transaminase were measured using VITROS 250 Microslide Technology (GMI Inc. Ramsey, MN).
10
Production and Purification of Cry4Ba Toxin
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Escherichia coli cells JM109 harboring the pMU388 plasmid encoding for Cry4Ba and its mutants R158A (12) , R158E, R158Q (13) , Y170A and Y170F (14) (link) were grown at 37℃ in Luria-Bertani medium containing 100 μg/ml ampicillin. Protein expression and production of toxin inclusions were performed as previously described (9) (link). Protoxin inclusions (130 kDa) were solubilized in 50 mM sodium carbonate (pH 10.0) and digested with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Sigma-Aldrich, St. Louis, MO), resulting in a 65 kDa of the activated toxin, which was subjected to protein purification by gel filtration chromatography equipped with a Superose 12 column (GE Healthcare, Uppsala, Sweden). The purified 65-kDa protein was resolved by (12% gel) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein bands of the 47 kDa and the 18-20 kDa were then recovered by electroelution.
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