The isolation trials were performed in an ÄKTA Avant system with UNICORN 6 software (GE Healthcare, Wauwatosa, WI, USA) at room temperature. All buffers were filtered through a 0.22 μm pore size membrane and ultrasonically degassed. Butyl-Sepharose 4FF and Octyl-Sepharose 6 FF (GE Healthcare, Wauwatosa, WI, USA) were used as HIC stationary phases. The hydrophobic matrices were packed according to the company guidelines (10 mL gel volume packed into an XK-16 glass column, GE Healthcare, Wauwatosa, WI, USA). The columns were equilibrated with the different tested concentrations of ammonium sulfate in Tris-HCl 10 mM, pH 7.8. LNCaP total protein extracts (500 µL with a protein concentration of ~12 mg/mL) were loaded onto the columns. Isocratic elution at 1.0 mL/min was performed by decreasing stepwise the ammonium sulfate concentration up to 0 M. The pH, pressure, conductivity, and 280 nm absorbance were continuously monitored throughout the entire chromatographic run. The fractions of interest were collected, desalted, concentrated, and stored at 4 °C to further purity and immunoreactivity analysis. The protein content of HIC fractions was measured by Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA).
Unicorn 6
Manufactured by GE Healthcare
Sourced in United States, Sweden
UNICORN™ 6.3 software is a comprehensive data management and analysis platform designed for use with GE Healthcare's life science laboratory equipment. The software enables users to collect, analyze, and manage data generated from various laboratory instruments. It provides a streamlined interface for data processing, report generation, and system administration.
Automatically generated - may contain errors
Lab products found in correlation
Kta avant system, by GE Healthcare (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Chemstation software, by Agilent Technologies (1 mentions)
Hitrap mabselect xtra column, by GE Healthcare (1 mentions)
Agilent 1100 hplc system, by Agilent Technologies (1 mentions)
Hitrap, by Cytiva (1 mentions)
Mabselect, by Cytiva (1 mentions)
Akta avant, by Cytiva (1 mentions)
Superdex 200 increase 10 300 gl column, by GE Healthcare (1 mentions)
Kta avant 150, by GE Healthcare (1 mentions)
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Superose 12 10 300 gl column, by GE Healthcare (1 mentions)
Kta pure system, by GE Healthcare (1 mentions)
Originpro 2016, by OriginLab (1 mentions)
Histrap ff crude, by GE Healthcare (1 mentions)
Vivaspin concentrator, by Sartorius (1 mentions)
7 protocols using unicorn 6
1
Hydrophobic Interaction Chromatography of LNCaP Proteins
2
Protein Purification using SEC and Affinity Chromatography
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Size exclusion chromatography (SEC) was carried out using an Agilent 1100 HPLC system with ChemStation software (Agilent Technologies UK Ltd., Berkshire, UK) with a TSK Gel 7 mm × 300 mm G3000SWXL column (Tosoh Bioscience LLC, Montgomeryville, USA). Affinity chromatography was carried out using an AKTA Avant 25 liquid chromatography system with Unicorn 6 software (GE Healthcare, Uppsala, Sweden), with a 1 mL HiTrap MabSelect Xtra column (GE Healthcare UK Ltd., Buckinghamshire, UK) attached.
3
Size Exclusion Chromatography of BSM
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A BSM solution (4.25 mg mL-1) was prepared with 20 mM PIPES, 300 mM NaCl buffer (working buffer), at pH 5, and filtered with a 0.45 μm Minisart® High Flow Hydrophobic PES syringe filter. SEC was conducted using a Superdex 200 Increase 10/300 GL column (GE Healthcare, USA) coupled to an ÄKTA™ avant 150 liquid chromatography system (GE Healthcare, USA) equipped with UV and conductivity/pH monitors. System operation and data gathering and analysis were performed using the UNICORN™ 6.3 software (GE Healthcare, USA). The column was loaded with 1 mL of BSM at a constant flow rate of 0.6 mL.min-1. Working buffer was used as eluent and the eluted fractions were collected for further analysis. Elution of BSM was monitored at 230, 260 and 280 nm.
4
Purification of Recombinant Proteins from Cell-Free Extracts
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Cell free extracts (CFEs) were prepared by resuspending harvested cell pellets in 20 mM potassium phosphate buffer, 500 mM NaCl, pH 7.4, conatining 10 mM imidazole for purification of recombinant protein. The prepared CFE was filtered through a 0.22 μm syringe filter prior to purification. Protein purification was conducted with prepacked, ready‐to‐use columns on an Äkta Pure 100 with a fraction collector F950 (Unicorn 6.3 software, GE Healthcare) by applying a concentration gradient of imidazole. For buffer exchange and removal of imidazole after purification, proteins were desalted using PD‐10 columns, previously equilibrated with desalting buffer (50 mM MES, pH 7.5, containing 10 mM MgCl2, 1 mM EDTA, and 1 mM DTT). Aliquots of the protein solution were shock frozen in liquid nitrogen and stored at −80 °C. Protein concentrations were determined with a Nanodrop 2000c spectrophotometer (Thermo Scientific).
5
Protein Size Exclusion Chromatography
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The size exclusion chromatography (SEC) protocol was as follows. Fifty μg of protein (at a concentration of 0.5 mg/mL, with or without heat stress; Table S1) was injected into an ÄKTA Pure system (GE Healthcare) and fractionated using a Superose 12 10/300 GL column (GE Healthcare) at 25 o C. The mobile phase consisted of 8 M urea, 150 mM NaCl, 50 mM sodium citrate, pH 3.0 at a flow rate of 0.5 mL/min. Running time was 40 min and UV detection at 280 nm was used to monitor the elution profile.
The calibration curve was performed using blue dextran, ferritin type 1, bovine serum albumin and cytochrome c as standards. The standards were all injected in triplicate starting from 0.5 mg/mL solutions. Data were treated in the UNICORN 6.3 software (GE Healthcare) and posterior analysis of the chromatograms was performed in OriginPro 2016 software (OriginLab).
The calibration curve was performed using blue dextran, ferritin type 1, bovine serum albumin and cytochrome c as standards. The standards were all injected in triplicate starting from 0.5 mg/mL solutions. Data were treated in the UNICORN 6.3 software (GE Healthcare) and posterior analysis of the chromatograms was performed in OriginPro 2016 software (OriginLab).
6
Purification of Recombinant hSCOMTVal108-6His
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The purification of the human recombinant SCOMTVal108-6His was performed according to the procedure described by Pedro et al. [30 (link)]. Briefly, chromatographic experiments were performed in Äkta Start system with UNICORN 6.1 software (GE Healthcare, Sweden). The chromatographic runs were performed at 20 °C on a HisTrapTM FF crude (5 mL) (GE Healthcare, Sweden). The column was equilibrated under the same conditions described by our research group in [30 (link)]. The resuspended pellet in the equilibrium buffer was applied onto the column using a sample pump at a flow rate of 0.5 mL min−1. Under the conditions described and after the elution of proteins that do not have any interaction with the column, the concentration of imidazole was increased in a step mode of 50, 70, 300, and 500 mM. All the elution steps were performed with 5 CVs at 1 mL min−1. Purified hSCOMTVal108-6His were pulled at 300 mM of imidazole, concentrated, and desalted with Vivaspin concentrators (10,000 MWCO) (Sartorius, Gottingen, Germany). The purified samples were stored at 4 °C until use for further assays.
7
Bispecific Antibody Purification by Ion Exchange
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Chromatography resins screened were packed to 20 ± 1.0 cm bed height in 1.0 cm ID columns. Columns were equilibrated with 2 CVs of 20 mM sodium phosphate, pH 7.2 before load application of bsAb C to 10 g FcFc + bsAb/L. Load material had been previously subjected to affinity capture as described above, and was 77% bispecific purity as per Eq. 1. Following loading, columns were washed with a proprietary wash buffer system and eluted in 40 mM acetate, 500 mM calcium chloride, pH 6–3 over 20 CV. All steps were performed at a 4 min residence time. UNICORN™ 6.1 software (GE Healthcare) was used for chromatographic analysis including calculation of chromatographic peak resolution (Rs see Eq. 2) assuming Gaussian peaks using the width at half height method (resolution algorithm 3).
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