For large preparations of hybridoma supernatant, antibodies were purified using a HiTrap MabSelect SuRe column (GE Healthcare Life Sciences, Cat# 11003494) on an AKTA Pure FPLC system (GE Healthcare Life Sciences). Supernatant was sterile filtered with a 0.2 μm filter and loaded on the column. After washing, antibodies were eluted with 100 mM glycine, 150 mM NaCl, pH 3.0. Eluate was immediately neutralized with 1 M Tris-base, pH 9.0. The UV trace was used to select and pool fractions containing antibody. Antibody was concentrated using Amicon Ultra-15 50 K centrifugal filter units (Millipore Sigma, Cat# UFC905024) and dialyzed into phosphate-buffered saline, pH 7.2. Antibodies were then sterile filtered with a 0.2 μm filter, protein concentration in each sample was determined by a bicinchoninic acid colorimetric assay (Fisher Cat# 23223 and 23224), using bovine serum albumin as a standard (Thermo Fisher Cat# 23210). Samples were run on a 15% SDS-PAGE gel and coomassie stained to ensure presence of heavy and light changes and protein purity. After purification from hybridoma supernatant, antibodies were frozen in 1 mL aliquots and stored at −20°C.
Hitrap mabselect sure column
Manufactured by GE Healthcare
Sourced in United States
The HiTrap MabSelect SuRe column is a lab equipment product designed for the affinity purification of monoclonal antibodies (mAbs). It features protein A ligand immobilized on a sepharose resin, providing a reliable and efficient method for capturing and purifying mAbs from cell culture supernatants or other samples.
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Lab products found in correlation
Expi293f cells, by Thermo Fisher Scientific (7 mentions)
Pierce fab preparation kit, by Thermo Fisher Scientific (4 mentions)
Hitrap mabselectsure columns, by GE Healthcare (4 mentions)
Superdex 200 increase 10 300 column, by GE Healthcare (4 mentions)
Akta pure fplc system, by GE Healthcare (2 mentions)
Anti ch1 column, by GE Healthcare (2 mentions)
0.45 μm pore size filter devices, by Merck Group (2 mentions)
Crystallized papain, by Merck Group (2 mentions)
Papain, by Merck Group (2 mentions)
Hitrap protein g hp column, by GE Healthcare (2 mentions)
Expifectamine 293 transfection kit, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Recombinant protein a, by GE Healthcare (1 mentions)
Ptwist cmv betaglobin wpre neo mammalian expression vector, by Twist Bioscience (1 mentions)
Gibco hybridoma sfm, by Thermo Fisher Scientific (1 mentions)
Desalting column, by GE Healthcare (1 mentions)
Kta start system, by GE Healthcare (1 mentions)
Pd miditrap g25 units, by GE Healthcare (1 mentions)
Vp dsc microcal llc equipment, by GE Healthcare (1 mentions)
Origin 7, by OriginLab (1 mentions)
28 protocols using hitrap mabselect sure column
1
Affinity Purification of Hybridoma Antibodies
2
Recombinant Anti-GDob1 IgG3 Allotype Production
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Two monoclonal recombinant anti-GDob1 IgG3 allotypes, G3m(g) and G3m(s), were produced in an HEK-293F FreeStyle cell line expression system (Life Technologies, Paisley, UK). IgG3m(s) was then purified with a HiTrap MabSelect SuRE column packed with recombinant Protein A (GE Healthcare), while IgG3m(g) was purified with a Protein G HiTrap HP column (GE Healthcare).
3
Monoclonal Antibody Purification Workflow
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HCCF was loaded onto a prepacked HiTrap® MabSelect™ SuRe™ column (1 mL, GE Healthcare, Little Chalfont, UK) with the pressure threshold set at 0.5 MPa. The column was equilibrated with 10 column volumes (CVs) of 20 mM sodium phosphate pH 6.8 at a flow rate of 1 mL/min and the sample then loaded at 2.5 mL/min and the flow‐through collected. The column was washed with 5 CVs of 20 mM sodium citrate, pH 5 and the mAb then eluted with 3 CVs of 20 mM sodium citrate, pH 3.5 and the eluate collected. For HCCF subjected to Protein A affinity chromatography only, samples were run in 5 × 10 mL lots through the column, and then all eluate fractions combined. For the experiment analyzing HCCF, Protein‐A eluate, and then subsequent post‐cation and anion exchange material, 150 mL of HCCF was subjected to Protein A affinity purification in 15 × 10 mL runs with the subsequent eluates combined after each step. The mAb containing fractions were then pooled for further downstream processing, with a sample of the pooled fractions retained for subsequent HCP analysis.
4
Recombinant Fab and IgG Production
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For the expression of recombinant rH7-243 and rH7-197 Fabs, the nucleotide sequences of antibody heavy and light chain antibody variable genes were codon optimized for mammalian expression and synthesized at Twist Biosciences. The resulting gene fragments were cloned directly at Twist Biosciences into the pTwist CMV BetaGlobin WPRE NEO mammalian expression vector (Twist Biosciences). MAb heavy/light chain constructs were used to transfect Expi293F cells (Thermo Fisher Scientific, A14528) transiently, and supernatants were harvested after culturing for 6 to 7 days. Recombinant Fabs were purified with Anti-CH1 CaptureSelect column (Cytiva). IgGs produced by the corresponding human mAb-secreting hybridoma cell lines in serum-free medium (Gibco Hybridoma-SFM, Thermo Fisher Scientific) were purified by affinity chromatography using HiTrap MabSelect SuRe column (GE Healthcare Life Sciences).
5
Recombinant Antibody Expression and Purification
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Chemicals were purchased from Sigma. Abs against RSV (clone B21M, containing F405L) and HIV envelope glycoprotein gp120 (clone b12, containing K409R) in human IgG1 and human IgG1 lacking hinge disulfides (C226S/C229S) were expressed transiently in Expi293F cells (Thermo Fisher, A14527) according to the manufacturer's protocol (46 (link), 47 (link)). Purification was performed using a HiTrap MabSelect SuRe column (GE Healthcare Life Sciences, 11–0034-94) according to the manufacturer's protocol, followed by immediate buffer exchange over a desalting column (GE Healthcare Life Sciences, 17–5087-01) into fresh phosphate-buffered saline (2.67 mm KCl, 1.47 mm KH2PO4, 138 mm NaCl, 8.06 mm Na2HPO4, pH 7.2). SDS-PAGE and analytical size-exclusion chromatography (SEC) were used to analyze proteins. Hydrophobic interaction chromatography was used to ensure that cFAE went to completion (>90% bsAb) for both IgG1WT and IgG1C→S.
6
Thermal Characterization of Purified IgGs
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The IgGs were purified by protein A chromatography using a 1-mL pre-packed HiTrap™ MabSelect SuRe™ column (GE Healthcare, no. 29-0491-04) on an ÄKTA start system (GE Healthcare) according to the manufacturer’s recommendations. IgG samples were re-buffered by PD MidiTrap G25 units (GE Healthcare, no. 17-0851-01) in 30 mM phosphate buffer, 150 mM NaCl, and pH 6 and adjusted to 2–3 μM. Heat energy uptake was measured by a VP-DSC MicroCal LLC equipment (GE Healthcare). The heat capacity was monitored between 20 and 100 °C applying a scan rate of 1 °C/min. The baseline correction was performed by subtraction of a re-scan of the unfolded protein and the result was fitted with the Origin 7.0 software (OriginLab, Northampton, MA). For fitting, a non-two-state unfolding model was applied.
7
Antibody Purification and Fab Preparation
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For antibody purification from hybridoma supernates, HiTrap MabSelectSure columns (GE Healthcare Life Sciences) were used to purify antibodies using the manufacturer's protocol. To obtain fragment antigen-binding (Fab) fragments, papain digestion was used (Pierce Fab Preparation Kit, Thermo Scientific). Fab fragments were purified by removing IgG and Fc contaminants using a HiTrap MabSelectSure column followed by purification with an anti-CH1 column (GE Healthcare Life Sciences).
8
Purification of Protein via Affinity Chromatography
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FectoPRO® transfection cell culture medium was centrifuged and filtered through a 0.22 μm filter to remove cells and debris, then loaded onto a HiTrap™ MabSelect SuRe™ column (GE Healthcare Life Sciences; Cat #11003494) on the AKTA Pure system (Cytiva) pre-equilibrated with 10 mM NaPO4 and 150 mM NaCl at pH 7.0. After loading, the column was washed with 10 column volumes of the same buffer. The protein was eluted with 100 mM NaOAc, pH 3.6, then immediately neutralized using 2 M Tris pH 8.0. The elution fractions were pooled and dialyzed into 10 mM Hepes and 150 mM NaCl at pH 7.4.
9
Purification of Human IgG3 Antibody
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IgG3 was purified from six serum samples (average donor age: 44.5 ± 9 years; three male and three female; details are listed in supplemental Table S1 ). The serum samples were collected from healthy donors with informed consent in compliance with the institutional ethical board. Venous blood was collected in a 9-ml Vacuette serum clot activator tube (Greiner BioOne, Kremsmünster, Austria) and incubated at room temperature for 30 min, followed by centrifugation for 15 min at 1800 g. The serum fraction was then collected and stored at −20 °C. IgG was isolated by running the serum over a HiTrap Protein G HP column (GE Healthcare, Buckinghamshire, UK) and eluted with 0.1 m glycine-HCl, pH 2.7. The eluate containing IgG was then applied to a HiTrap MabSelect SuRE column packed with recombinant Protein A (GE Healthcare), and the IgG3-containing flow-through was concentrated using an Amicon Ultra-15 centrifugal filter device 10 kDa (Merck Millipore, Darmstadt, Germany) and dialyzed against PBS using a Slide-A-Lizer Dialysis Cassette, 10K MWCO (Dionex/Thermo Scientific, Sunnyvale, CA).
10
Affinity Purification and Protein Digestion
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HiTrap Mabselect SuRe column was purchased from GE Healthcare (Chicago, IL, USA) and C18 trap column was purchased from Harvard Apparatus (Holliston, MA, USA). Trypsin for protein digestion was purchased from Promega (Madison, WI, USA). Phosphate buffered saline (PBS), 1,4-dithiothreitol (DTT), iodoacetamide (IAA), trifluoroacetic acid (TFA), ammonium bicarbonate (ABC), and formic acid (FA) were purchased from Sigma Aldrich (St. Louis, MO, USA). HPLC-grade water and acetonitrile (ACN) were purchased from J.T. Baker (Phillipsburg, NJ, USA). CHO-k1 cells were purchased from Sigma Aldrich.
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