Divalent scFvF7-Fc Ab was produced through transient transfection of CHO DG44 cells followed by incubation for 6 days in ProCHO5 serum free medium (Lonza). The scFvF7-Fc Ab was purified from the cell medium using a protein A column (Thermo Fisher) with Abs eluted with 100 mM glycine buffer pH 3.0 into a 1 M Tris-HCl eluate at pH 8.0. Abs were dialyzed twice against PBS, concentrated using a JumboSep centrifuge filter with 3 kDa molecular weight cut-off (Pall Laboratories), and sterile filtered with Millex GP 0.22 μm pore size (Millipore). Prior to evaluation of scFvF7-Fc in the in vitro functional recovery assays, Abs were tested with the FDA-licensed Endosafe-PTS kit (Charles River Laboratory) which showed < 5 EU of endotoxins per mg of protein.
Protein a column
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Protein A column is a chromatography column used for the purification of antibodies and other proteins that bind to Protein A. It utilizes Protein A, a bacterial surface protein, immobilized on a solid support matrix to selectively capture and purify target proteins from complex mixtures.
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Lab products found in correlation
Nanodrop spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Pei max 40000, by Thermo Fisher Scientific (2 mentions)
Histidine tagged protein purification resin, by R&D Systems (2 mentions)
Amicon ultra 15 centrifugal filter unit 50 k, by Merck Group (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Procho5 serum free medium, by Lonza (1 mentions)
Endosafe pts kit, by Charles River Laboratories (1 mentions)
Millex gp, by Merck Group (1 mentions)
Polyjet reagent, by SignaGen (1 mentions)
Sanger sequencing, by Source Bioscience (1 mentions)
Expi293f cells, by Thermo Fisher Scientific (1 mentions)
One shot top10 e coli cells, by Thermo Fisher Scientific (1 mentions)
Igg elution buffer, by Thermo Fisher Scientific (1 mentions)
Expi293 cell, by Thermo Fisher Scientific (1 mentions)
Protein l column, by Thermo Fisher Scientific (1 mentions)
Binding buffer, by Thermo Fisher Scientific (1 mentions)
Elution buffer, by Thermo Fisher Scientific (1 mentions)
Tris hcl, by Avantor (1 mentions)
28 protocols using protein a column
1
Endotoxin-free scFvF7-Fc Antibody Production
2
Trimerization of HA-Fc Fusion Proteins
3
Cloning and Expression of Antibody Variable Regions
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Heavy and light chain variable regions were cloned in pVitro-hygro-1 (Invivogen) using polymerase incomplete primer extension (PIPE) cloning as previously described (25 (link)). In brief, pVitro-hygro-1, containing pre-cloned human antibody heavy and light constant region cassettes (gamma 1/kappa) was used as a template in two separate PIPE PCR reactions to amplify two linear plasmid fragments with partially single-stranded 5″ ends. Similarly, the Ig heavy and light chain variable regions were PIPE PCR-amplified, using the pCR-Blunt constructs, generated previously, as PCR templates. Next, the PCR products were diluted four times with ddH2O and mixed in a ratio of 1:1:1:1, incubated at RT for 1 h, and 10 µl of the mixture were used to transform Top10 OneShot™ E. coli cells (Thermo Fisher Scientific). Successful cloning was confirmed by Sanger sequencing (Source BioScience). MOv18 IgG1/k was expressed transiently in Expi293F™ cells (Thermo Fisher Scientific), as described in Ref. (26 (link)). Antibodies, secreted in the Expi293F™ culture supernatant, were purified using a Protein A column (Thermo Fisher Scientific) and stored in PBS at 4°C.
4
Generation and Characterization of Anti-CHI3L1 Antibody
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The murine monoclonal anti-CHI3L1 antibody (FRG) was generated using peptide antigen (amino acid 223–234 of human CHI3L1) as immunogen through Abmart Inc (Berkeley Heights, NJ). This monoclonal antibody specifically detects both human and mouse CHI3L1 with high affinity (kd ≈ 1.1 × 10–9). HEK-293T cells were transfected with the FRG construct using Lipofectamine 3000 (Invitrogen, # L3000015). Supernatant was collected for 7 days, and the antibody was purified using a protein A column (Thermo Fisher Scientific, #89960). Ligand-binding affinity and sensitivity were assessed using ELISA techniques.
5
Recombinant SARS-CoV-2 Antibody Production
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Codon-optimized antibody heavy-chain and light-chain sequences of CB6 and LY-Cov1404 were synthesized and cloned into a VRC8400-based IgG1 vector and co-expressed by transient transfection to Expi293 cells (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s recommendation. Briefly, 50 μg plasmid-encoding heavy-chain genes and 50 μg plasmid-encoding light-chain genes were mixed with 300 μL of Turbo293 transfection reagent (SPEED BioSystems, Gaithersburg, MD, USA) for 15 min, added to 100 mL of cells at a concentration of 2.5 × 106/mL, and incubated in a shaker incubator at 120 rpm and 37 °C under 9% CO2. On days 1 and 3 post transfection, an enriched feed medium, AbBooster Antibody Expression Enhancer for suspension cells (ABI Scientific, Sterling, VA, USA), was added into the culture at a 10% culture volume. After 5 days post transfection, cell culture supernatant was harvested and loaded onto a Protein A column (Thermo Fisher Scientific, Waltham, MA, USA). The antibody was eluted using IgG Elution Buffer (Thermo Fisher Scientific, Waltham, MA, USA) and brought to neutral pH (7.0) with 1 M Tris-HCl (pH 8.0). Eluted antibodies were dialyzed against PBS overnight before use.
6
Affinity Purification of Ig Antibodies
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Individual or pooled plasma samples (2 ml/sample) were applied to a Protein L column (cat # 89963, Thermofisher, Waltham, MA) per manufacture’s guidelines, incubated for 1h at room temperature, washed 3 times with 2 ml of phosphate buffered saline (PBS, pH7.0) and Ig eluted from the column with 0.1M glycine in 1 ml (pH 2-3) (cat # 21004, Thermofisher). The flow through and eluent were neutralized with 1M Tris-HCl (pH 8) (cat # 15568025, Thermofisher). The buffer was exchanged with PBS and samples concentrated using a 30,000 KD molecular weight Protein Concentrator (cat # 88522, Thermo Fisher) and resuspended in 1 ml total volume. The Ig-enriched samples were then either incubated with lectin-gD or lectin-gB agarose column for 1 hour, bound Ig eluted with 0.1M glycine, neutralized to pH 7 with 1M Tris-HCl, buffer exchanged and concentrated as above. Alternatively, the Protein L eluent was applied to a Protein A column (cat # 20356, Thermofisher), which binds human IgG1, IgG2 and IgG4 but not IgG3. A subset of samples was sequentially incubated with a gD or gB lectin agarose column followed by a Protein A column to enrich for IgG1 or IgG3 specific anti-gD and gB. After concentration, the total protein was quantified by nanodrop and all samples were diluted to a final concentration of 0.5 mg/ml for functional assays.
7
Soluble SARS-CoV-2 Spike Protein Production
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The pcDNA3.1 plasmids encoding S and S-Fc were transfected into CHO cells using PEI MAX 40000 (Fisher Scientific, Cat# NC1038561). Stable cell lines were selected and maintained under G418 (1 mg/ml). Immunofluorescence assay, SDS-PAGE, and Western blotting analysis determined the expression and secretion of the S or S-Fc fusion proteins. We produced the soluble S or S-Fc proteins by culturing CHO cells in a complete medium containing 5% FBS with ultra-low IgG. The proteins were captured by Protein A column (ThermoFisher Scientific, Cat# 20356) for the S-Fc protein or Histidine-tagged Protein Purification Resin (R&D Systems, Cat # IP999) for the S protein, eluted with 0.1 M Glycine (pH 2.5), and neutralized with 1 M Tris-HCl (pH 8.0). Glycine and Tris-HCl in the protein solution were replaced with PBS three times using centrifugation with Amicon Ultra-15 Centrifugal Filter Unit (50 K) (Millipore, Cat# UFC905024). Protein concentrations were determined using a NanoDrop spectrophotometer (Thermo Scientific).
8
Purification of Rabbit IgG Antibody
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IgG from rabbit serum was purified by affinity chromatography using a protein A column (Thermo Scientific). Serum was diluted 1:1 in binding buffer (PBS: 10 mM Na P , 150 mM NaCl, pH 7.2) before applied to the column. Bound proteins were eluted with elution buffer (0.1 M glycine pH 2.5). The IgG fraction was dialyzed in PBS for 24 h at 4 °C and concentrated with an Amicon Ultra centrifugal filter unit tube (Sigma Aldrich); finally, protein concentration was determined by the Bradford method. Purified anti- SPCB-NF antibody was used in all experiments.
9
Affinity-Based Antibody Purification
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Purification employed a protein A column for recombinant antibodies and a protein G column for hybridoma antibodies (ThermoFisher Scientific, Waltham, MA). Cellular supernatants were added at 1:10 v/v of the binding buffer appropriate for the column (ThermoFisher Scientific, Waltham, MA) to ensure proper ionic and pH conditions for later affinity matrix-binding. The insoluble precipitates were removed by centrifugation at 9,500 rpm for 10 mins at 4°C. The appropriate column was utilized according to the manufacturer’s recommendations for equilibration and sample loading to purify the Ig. The column was washed with 5 column volumes (CVs) of binding buffer prior to stepwise acid washes (2 CVs in each pH buffer, 8.5, 6.5, 5.5) followed by Elution buffer (ThermoFisher Scientific, Waltham, MA) and neutralized using 1/10 (v/v) of 1M Tris/HCl, pH 8.0 (VWR, Radnor, PA). Protein dye staining and gel electrophoresis were employed to identify Ig within collected fractions. The final Ig-yield was determined by BCA protein assay (ThermoFisher Scientific, Waltham, MA)
10
Recombinant SARS-CoV-2 Spike Protein Production
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The pcDNA3.1 plasmids encoding S and S-Fc were transfected into CHO cells using PEI MAX 40000 (Fisher Scientific, Cat# NC1038561). Stable cell lines were selected and maintained under G418 (1 mg/ml). Expression and secretion of the S or S-Fc fusion proteins were determined by immunofluorescence assay, SDS-PAGE, and Western blotting analysis. We produced the soluble S or S-Fc proteins by culturing CHO cells in a complete medium containing 5% FBS with ultra-low IgG. The proteins were captured by Protein A column (ThermoFisher Scientific, Cat# 20356) for the S-Fc protein or Histidine-tagged Protein Purification Resin (R&D Systems, Cat # IP999) for the S protein, eluted with 0.1M Glycine (pH 2.5), and neutralized with 1M Tris-HCl (pH8.0). Glycine and Tris-HCl in the protein solution were replaced with PBS three times using centrifugation with Amicon Ultra-15 Centrifugal Filter Unit (50K) (Millipore, Cat# UFC905024). Protein concentrations were determined using a NanoDrop spectrophotometer (Thermo Scientific).
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