The four different 2C1-Fc fusion proteins were cloned and expressed by the CRO Evitria AG (Schlieren, Switzerland). For purification, 250 ml of cell culture supernatant was applied onto a Mabselect SuRe column (GE Healthcare) using an ÄKTA purifier system (GE Healthcare). The column was washed with 15 column volumes of PBS, pH 7.4, and the protein was eluted using 0.1 m glycine, pH 2.8, collecting 1-ml fractions. After elution, pH was adjusted with 1 m Tris, pH 9. The OD of the fractions was determined using an Infinite M200 pro reader and a NanoQuant plateTM (Tecan). The two fractions showing the highest OD were loaded onto an ÄKTA purifier. Preparative size exclusion was performed using a Superdex 200 10/300 GL column (GE Healthcare), and the storage buffer was exchanged to phosphate-buffered saline (PBS, pH 7.4, Invitrogen). The four highest OD fractions were combined and used for further studies. Protein size, purity, and aggregation state was analyzed using an ÄKTA purifier system (GE Healthcare).
Kta purifier system
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Sweden, Germany, Japan
The ÄKTA purifier system is a versatile liquid chromatography platform designed for protein purification. It provides precise control and monitoring of key parameters such as flow rate, pressure, and UV absorbance to enable efficient separation and purification of biomolecules.
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Lab products found in correlation
Superdex 200 10 300 gl column, by GE Healthcare (11 mentions)
Scientz iid, by Ningbo Scientz Biotechnology (6 mentions)
Superdex 75 10 300 gl column, by GE Healthcare (5 mentions)
Dawn 8 8 angle light scattering detector, by Wyatt Technology (4 mentions)
Astra6 software package, by Wyatt Technology (4 mentions)
Biochrom 30 series amino acid analyzer, by Harvard Bioscience (3 mentions)
Pepclean c18 spin column, by Thermo Fisher Scientific (3 mentions)
Optilab t rex refractometer with extended range, by Wyatt Technology (3 mentions)
Spherisorb column, by Waters Corporation (2 mentions)
Complete edta free protease inhibitor cocktail, by Roche (2 mentions)
Monoq 10 100 gl, by GE Healthcare (2 mentions)
Superdex 200 5 150 gl column, by GE Healthcare (2 mentions)
Prism 5, by GraphPad (2 mentions)
Bagmixer 400 p, by Interscience (2 mentions)
Superdex s200 hr 10 300, by GE Healthcare (2 mentions)
Superdex 200 increase 10 300 gl column, by GE Healthcare (2 mentions)
Prescission protease, by GE Healthcare (2 mentions)
Superdex 200 gel filtration column, by GE Healthcare (2 mentions)
Ni nta affinity column, by Thermo Fisher Scientific (2 mentions)
Superose 6 increase 10 300 gl column, by GE Healthcare (2 mentions)
136 protocols using kta purifier system
1
Purification of 2C1-Fc Fusion Proteins
2
Quantification of Carbohydrates and Organic Acids in Fermented and Unfermented Samples
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Equal volumes of perchloric acid (5%, vol/vol) were added to fermented and unfermented CP aliquots as precipitating agent. The suspension was kept at 4°C overnight, centrifuged at 10,000 × g, 10 min, and filtered through a Millex-HA 0.22-mm pore size filter (Millipore Co., Bedford, MA). The concentration of glucose, fructose and sucrose was determined through HPLC analysis, using an ÄKTA Purifier system (GE Healthcare) equipped with a Spherisorb column (Waters, Millford, USA) and a Perkin Elmer 200a refractive index detector. Elution was at 32°C with a flow rate of 1 ml/min, using acetonitrile 80% as the mobile phase [25 ]. Organic acids were determined by HPLC, using an ÄKTA Purifier system (GE Healthcare) equipped with an Aminex HPX-87H column (ion exclusion, Biorad) and a UV detector operating at 210 nm. Elution was at 60°C with a flow rate of 0.6 ml/min, using 10 mM H2SO4 as the mobile phase [26 (link)]. Peaks were identified by comparing elution times and spiking samples with known quantities of standard solutions of acetic and lactic acid. Total and individual free amino acids were analyzed by a Biochrom 30 series Amino Acid Analyzer (Biochrom Ltd., Cambridge Science Park, England), with a Na-cation-exchange column (20 by 0.46 cm inner diameter) as described by Rizzello et al. [27 ].
3
Peptide Fractionation by Strong Cation Exchange
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Twelve labeled peptide extracts were fractionated by SCX using an ÄKTA purifier system (GE Healthcare, Piscataway, NJ, USA). Each sample was dried in a vacuum centrifuge and reconstituted with solvent A (10 mM KH2PO4 in 25% [v/v] acetonitrile, pH 3.0). The resulting sample was then loaded onto a Polysulfoethyl A column (PolyLC Inc., Columbia, Maryland, USA) with a 4.6-mm inner diameter and length of 100 mm. The resulting peptides were first eluted at a 1 mL min−1 flow rate with a step gradient as follows: solvent A for 40 min, 0% solvent B (500 mM KCl, 10 mM KH2PO4 in 25% [v/v] acetonitrile, pH 3.0) for 25 min, 0–10% solvent B for 7 min, 10%−20% solvent B for 10 min, 20%−45% solvent B for 5 min, 45%−100% solvent B for 5 min, and 100% solvent B for 8 min. The resulting eluent was monitored by checking the absorbance at 214 nm. Fractions were collected at 1 min, dried in a vacuum centrifuge, and dissolved with 0.1% (v/v) trifluoroacetic acid. Fraction collections were desalted with SPE C18 Cartridges (Empore™ standard density C18 cartridges; bed I.D. 7 mm, 3 mL volume; Sigma, St. Louis, MO, USA), lyophilized, and redissolved in buffer A (0.1% [v/v] formic acid in water).
4
Heme-Loaded Human Serum Albumin Preparation
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Hemin, biliverdin, and protoporphyrin were dissolved in 1 M NaOH (3 × 10−7 M) and incubated with 10 mg/ml HSA for 1 h at room temperature. The protein was dialyzed for 48 h against 1× PBS, changing the buffer after 24 h, to remove non-bound hemin. HSA-heme, HSA-biliverdin and HSA-protoporphyrin were then sterile filtered. The loading of HSA with hemin was analyzed after filtration by measuring the absorbance spectra. The ratio of HSA: heme was 1: 1,5 ± 0,36 (n = 8). At a protein concentration of 200 µg/ml, HSA-heme (1,8 × 1018 protein molecules, 251 µg/ml iron) and transferrin (1,5 × 1015 protein molecules, 0.3 µg/ml iron) carry 2,7 × 1018 and 3 × 1015 iron molecules, respectively.
HSA was labeled with Alexa Fluor 488 (NHS; HSA-AF488) dye according to the manufacturer´s instructions (Thermo Fisher Scientific Inc., Waltham, US). After labeling a purification run using a S200 column (Superdex 200, 10/300 GL, GE Healthcare Life Sciences) on an ÄKTA purifier system (GE Healthcare Life Sciences, Pittsburgh, US) was performed. Protein was concentrated and set to a concentration of 12 mg/ml.
HSA was labeled with Alexa Fluor 488 (NHS; HSA-AF488) dye according to the manufacturer´s instructions (Thermo Fisher Scientific Inc., Waltham, US). After labeling a purification run using a S200 column (Superdex 200, 10/300 GL, GE Healthcare Life Sciences) on an ÄKTA purifier system (GE Healthcare Life Sciences, Pittsburgh, US) was performed. Protein was concentrated and set to a concentration of 12 mg/ml.
5
Venom Fractionation by RP-HPLC
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Five hundred micrograms of lyophilized individual and pooled venoms were dissolved in 200 μL of 0.1% trifluoroacetic acid (TFA (Sigma, 302031); solution A), centrifuged at 13,000 x g for 15 min, and separated by RP-HPLC using a Teknokroma Europa Protein 300 C18 column (0.46 cm × 25 cm, 5 mm particle size, 300 Å pore size) and an Äkta Purifier system (GE Healthcare). Elution was carried out at 1 mL/min by applying a gradient toward solution B (95% acetonitrile (Sigma, 34851) containing 0.1% TFA), according to Gay et al. [64 (link)] with some modifications: 5% B for 5 min, 5–25% B for 10 min, 25–45% B for 60 min, 45–70% B for 10 min, 70–100% B for 10 min, and 100% B for 10 min.
6
Extraction and Analysis of Beverage Components
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Water/salt-soluble extracts from beverages were prepared following the method of Weiss, Vogelmeier & Gorg [35 (link)]. An aliquot of beverage (containing 1 g of flour) was diluted with 4 mL of Tris-HCL (pH 8.8), held at 4 °C for 1 h, vortexing at 15-min intervals, and centrifuged at 20,000× g for 20 min. The supernatant, containing the water/salt-soluble fraction, was filtered through a Millex-HA 0.22-µm pore size filter (Millipore Co., Bedford, MA, USA) and used for analysis. Organic acids contained in the water/salt-soluble extracts were determined by High Performance Liquid Chromatography (HPLC) using an ÄKTA Purifier system (GE Healthcare, Buckinghmshire, UK) equipped with an Aminex HPX-87H column (ion exclusion, Biorad, Richmond, CA, USA), and a UV detector operating at 210 nm [36 (link)]. Total and individual free amino acids were analyzed by a Biochrom 30 series Amino Acid Analyzer (Biochrom Ltd., Cambridge Science Park, UK) with a Na-cation-exchange column (20 by 0.46 cm internal diameter) as described by Rizzello, Nionelli, Coda, De Angelis & Gobbetti [37 (link)].
7
Validating Neutralizing Antibody Efficiency
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Neutralizing antibodies against murine CD4 and CD8 are produced from hybridomas TIB-207 and TIB-105, respectively. Antibodies were isolated and purified in our laboratory by affinity chromatography with ÄKTA Purifier system (GE Healthcare Life Sciences).
To validate their in vivo efficiency, these antibodies were injected intraperitoneally in C57BL/6 wt mice daily for three consecutives days at 200 μg for each mouse. On day 4, lymph nodes and spleen of each mouse were recovered and crashed in a manual manner through a Cell Strainer (Falcon). Then, extracted cells were analyzed for CD4+ and CD8+ population by flow cytometry.
For tumor growth experiments, anti-CD4 and anti-CD8 neutralizing antibodies were injected intraperitoneally in C57BL/6 wt mice at day 0, 1, 2, 4, 7, and 11 after tumor inoculation at 200 μg for each mouse.
To validate their in vivo efficiency, these antibodies were injected intraperitoneally in C57BL/6 wt mice daily for three consecutives days at 200 μg for each mouse. On day 4, lymph nodes and spleen of each mouse were recovered and crashed in a manual manner through a Cell Strainer (Falcon). Then, extracted cells were analyzed for CD4+ and CD8+ population by flow cytometry.
For tumor growth experiments, anti-CD4 and anti-CD8 neutralizing antibodies were injected intraperitoneally in C57BL/6 wt mice at day 0, 1, 2, 4, 7, and 11 after tumor inoculation at 200 μg for each mouse.
8
Purification of Phospholipase by HPLC
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Fraction D3 obtained in the chromatographic step on DEAE Sepharose was subjected to a C18 reverse phase column (4.6 mm ID × 25 cm, CLC-ODS, Shimadzu, Japan) using ÄKTA™ purifier system (GE Healthcare, USA). The column had been previously equilibrated with a solution of 0.1 % trifluoroacetic acid (TFA) (solvent A), and about 10 mg of fraction D3 was diluted in the same solvent and applied to the system using a 500 μL loop. Elution was performed at a flow rate of 0.5 mL/minute with a linear concentration gradient solution containing 70 % acetonitrile and 0.1 % TFA (Solvent B): 0-100 % solvent B in ten column volumes. All eluted fractions were assessed for their phospholipase activity and on SDS-PAGE, as described below. The fraction that showed phospholipase activity was pooled, lyophilized and rechromatographed in the same column, this time using a segmented concentration gradient of 0-60 % solvent B in three column volumes, 60-80 % in five column volumes, and 80-100 % in one column volume.
The purity level of the fraction D4 after Vivaspin® 20 was also evaluated in this reverse phase column using a linear concentration gradient of 0-100 % solvent B in five column volumes.
The purity level of the fraction D4 after Vivaspin® 20 was also evaluated in this reverse phase column using a linear concentration gradient of 0-100 % solvent B in five column volumes.
9
Fractionation of B. atrox Venom
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Chromatographic fractionation of B. atrox venom to obtain the toxins of interest began with a molecular exclusion step on Sephacryl S-200, followed by anion exchange chromatography on DEAE Sepharose. The fraction containing the metalloprotease (MP) was then ultrafiltered in a concentrator tube with membrane of MWCO 3,000, Vivaspin® 20 (Sartorius, Germany), while the fraction containing the phospholipase A2 (PLA2) was subjected to a C18 reverse phase column using ÄKTA™ purifier system. The classical chromatography resins as well as the reverse phase column and the ÄKTA™ system were obtained from GE Healthcare (USA).
The absorbance of the chromatographic fractions were measured at a wavelength of 280 nm, using a spectrophotometer Thermo Scientific™ GENESYS 10 UV (Thermo Fisher Scientific, Inc., USA) or the UNICORN™ 5.11 software for the ÄKTA™ purifier system (GE Healthcare, USA). Then, data were plotted on graphs using Origin 8 software for the obtainment and analysis of the chromatographic profiles.
The absorbance of the chromatographic fractions were measured at a wavelength of 280 nm, using a spectrophotometer Thermo Scientific™ GENESYS 10 UV (Thermo Fisher Scientific, Inc., USA) or the UNICORN™ 5.11 software for the ÄKTA™ purifier system (GE Healthcare, USA). Then, data were plotted on graphs using Origin 8 software for the obtainment and analysis of the chromatographic profiles.
10
ITC Analysis of BSA-SOCL Binding
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ITC experiments were carried out using an Auto-iTC200 system (MicroCal, Malvern-Panalytical, Malvern, England, UK), as described, in detail, in [48 (link),53 (link)]. The BSA was, first, purified by size-exclusion chromatography using a Superdex 200 10/30 HR column connected to an ÄKTA-purifier system (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The Superdex 200 10/30 HR column was calibrated with the standard proteins of the gel filtration high molecular weight (HMW) kit supplied by GE Healthcare. A 12.5 μM solution of monomeric BSA in PBS pH 7.4 was titrated at 25 °C with a 300 μM SOCL solution. Sets of 19 injections of 2 μL were performed. The resulting heats were integrated and normalized by the amount of ligand added per injection. The heat of SOCL dilution was accounted for by introducing an additional adjustable parameter in the analysis. Further details about the ITC analysis were given in Section 2 .
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