Proteins were expressed in Expi293 cells by transient transfection. IgGs and a previously described human ACE2-Fc construct11 (link) were purified from cell supernatants using MabSelect SURE columns (Cytiva), and His-tagged Fabs were isolated from cell supernatants using Ni-NTA columns (Qiagen). IgGs, ACE2-Fc, and Fabs were further purified by SEC using a HiLoad 16/600 Superdex 200 column (Cytiva). Purified proteins were concentrated using a 100 kDa and 30 kDa cutoff concentrator (EMD Millipore), respectively, to 10 to 15 mg/mL, and final concentrated proteins were stored at 4 °C until use. 6P versions25 (link) of soluble SARS-CoV-2 WA1 and SARS-CoV-2 Omicron BA.1 spike trimers were isolated from cell supernatants using a pre-packed Ni-NTA column (Cytiva). Eluents from Ni-NTA purifications were subjected to SEC using a HiLoad Superdex 200 16/600 column followed by a Superose 6 10/300 (Cytiva) column. Peak fractions were pooled and concentrated to ∼6 mg/ml, flash frozen in 50 μL aliquots, and stored at -80 °C until use.
Mabselect sure column
Manufactured by Cytiva
Sourced in United States
The MabSelect SuRe column is a protein A affinity chromatography resin designed for efficient and reliable capture of monoclonal antibodies from complex samples. The resin features a highly stable and durable matrix that can withstand repeated cleaning and sanitization cycles, ensuring consistent performance over extended use.
Automatically generated - may contain errors
Lab products found in correlation
Expifectamine transfection kit, by Thermo Fisher Scientific (4 mentions)
Expi293f cells, by Thermo Fisher Scientific (3 mentions)
Hiload 16 600 superdex 200 column, by Cytiva (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Ni nta column, by Qiagen (1 mentions)
Ni nta column, by Cytiva (1 mentions)
Superose 6 10 300, by Cytiva (1 mentions)
Hitrap protein a column, by Cytiva (1 mentions)
Expi293 expression system, by Thermo Fisher Scientific (1 mentions)
Kta start, by Cytiva (1 mentions)
Unicorn start 1, by Cytiva (1 mentions)
Hiload 26 600 superdex 75 pg column, by Cytiva (1 mentions)
8 protocols using mabselect sure column
1
Purification of SARS-CoV-2 Spike Proteins and Antibodies
2
Expression and Purification of Cysteine Mutants
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Expression of single unpaired cysteine mutants was performed in Expi293F suspension cells. The cells were transiently transfected with mutated DNA plasmid using Expi293 expression system (ThermoFisher Scientific, Waltham, MA, USA, Catalog No. A14635) in 96-well plates at 1.25 × 106 cells/0.5 mL/well, according to the manufacturer’s instructions. Each mutant was expressed in multiple wells (6 or 10 wells) to harvest 3 or 5 mL of conditioned media for Protein A purification. The 3 mL harvests were applied to 80 µL PhyNexus tips (San Jose, CA, USA) on a Hamilton Microlab STAR Liquid Handling System (Hamilton, ON, Canada). The proteins were captured, washed, and eluted with proprietary PhyNexus buffers (San Jose, CA, USA) . The 5 mL harvests were purified using 1-mL HiTrap protein A columns (Cytiva, Marlborough, MA, USA) on a Protein Maker (Protein BioSolutions, Gaithersburg, MD, USA). Double cysteine mutants were also expressed from Expi293 cells at 10 mL scale in 50 mL bioreactor tubes. They were purified using 1-mL MabSelect Sure columns (Cytiva, Marlborough, MA, USA) on a Protein Maker and buffer-exchanged into PBS using Amicon-15 filters (MWCO 10 kDa).
3
Production and Purification of SARS-CoV-2 Antibodies
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The variable region of the heavy and light chains of CR302254 (link), S30955 (link), and S2P656 (link) were cloned into the TGEX-HC and TGEX-LC vectors (Antibody Design Labs), respectively, according to the manufacturer’s protocol. ACE2 (residues 1–615) was cloned into TGEX-HC. The plasmids were expressed in Expi293F cells (ThermoFisher Scientific) using the ExpiFectamine Transfection Kit (Thermo Fisher Scientific) and transfected according to the manufacturer’s protocol. After incubation for 6 days at 37 °C for, the cells were centrifuged at 6000 × g for 15 min. The supernatant was diluted in MabSelect Binding Buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.2) and passed through a 1-mL MabSelect SuRe column (Cytiva) connected to an ÄKTA Start. The column was washed, and the proteins were eluted according to the manufacturer’s protocol. Fractions containing the protein were collected and dialyzed into PBS, then the concentration of antibodies and ACE2-Fc was measured using the BCA assay.
4
Cloning and Expression of CR3022 and ACE2
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The variable regions of the heavy and light chains of CR302217 (link) were cloned into the TGEX-HC and TGEX-LC vectors (Antibody Design Labs), respectively, according to the manufacturer’s protocol. Likewise, ACE2 (residues 1–615) was cloned into TGEX-HC. The DNA was then transfected into Expi293F cells (Thermo Fisher Scientific) by using the ExpiFectamine Transfection Kit (Thermo Fisher Scientific) following the provided protocol, and the cells were incubated in a humidified incubator at 37 °C and 8% CO2 for 5 days. The cells were then centrifuged at 5500 × g for 20 min. The supernatant media was diluted twofold in PBS and run through a 1-mL MabSelect SuRe column (Cytiva) connected to an ÄKTA start (Cytiva) and controlled by Unicorn start 1.0 software (Cytiva) according to the manufacturer’s operation manual to purify the proteins. CR3022 and ACE2-Fc were quantified by using the BCA assay (Thermo Scientific).
5
Expression and Purification of SARS-CoV-2 Antibodies
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The variable region of the heavy and light chains of CR302236 (link), S30937 (link), 0304–3H338 (link), and S2P639 (link) were cloned into the TGEX-HC and TGEX-LC vectors (Antibody Design Labs), respectively, according to the manufacturer’s protocol. ACE2 (residues 1 – 615) was cloned into TGEX-HC. The plasmids were expressed in Expi293F cells (ThermoFisher Scientific) using the ExpiFectamine Transfection Kit (Thermo Fisher Scientific) and transfected according to the manufacturer’s protocol. After incubation for 6 days at 37 °C for, the cells were centrifuged at 6000 ×g for 15 minutes. The supernatant was diluted in MabSelect Binding Buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.2) and passed through a 1-mL MabSelect SuRe column (Cytiva) connected to an ÄKTA Start. The column was washed and the proteins were eluted according to the manufacturer’s protocol. Fractions containing the protein were collected and dialyzed into PBS, then the concentration of antibodies and ACE2-Fc was measured using the BCA assay.
6
Protein Purification Using Affinity and SEC
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CM was filtered using a 0.45-μm membrane and loaded onto an affinity column coupled with a SEC column via the sample pump of AKTA (Cytiva). For Fc-tagged proteins, a 5–10 mL mAb Select Sure column (cat. no. 11003495, Cytiva) pre-equilibrated with TBS (20 mM Tris, 150 mM NaCl, pH 7.5) was used for capture. Columns were washed with 8 column volumes (CVs) of TBS and bound protein was eluted with 0.5% acetic acid, 150 mM NaCl, pH 3.5 into a 27-mL loop which was subsequently injected onto HiLoad Superdex 200 pg preparative SEC (cat. no. 28989336, Cytiva) with a running buffer of HBS (30 mM HEPES, 150 mM NaCl, pH 7.6). Peaks were pooled and filtered through a 0.22-μm Posidyne syringe filter (cat. no. 4908, Pall Corporation) before storing in -80°C. Protein concentration was determined using Nanodrop (Thermo Fisher Scientific). For His-tagged proteins, 5–10 mL of HisTrap excel column (cat. no. 17371206, Cytiva) pre-equilibrated with TBS with 20 mM imidazole was used. After loading, column was washed with the equilibration buffer for 15 CV. Bound protein was eluted with 20 mM Tris, 150 mM NaCl, 400 mM imidazole, pH 7.5 into a 27-mL loop and further purified by SEC. Sample pump, affinity and SEC columns were cleaned with 0.2 M NaOH and re-equilibrated with TBS or HBS after each run.
7
Recombinant 0304-3H3 Antibody Production
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The genes encoding the variable regions of the heavy chain and light chain of the 0304-3H3 antibody21 (link) were cloned into the TGEX-HC and TGEX-LC vectors (Antibody Design Labs), respectively, by Gene Universal, Inc. (Newark, DE). The plasmids were co-transfected in a 2:1 light chain to heavy chain ratio into Expi293F cells (RRID: CVCL_D615) using the ExpiFectamine Transfection Kit (Thermo Fisher Scientific) and associated protocol. After a 4-day incubation, the culture was centrifuged at 5,500×g for 20 min. The supernatant was diluted in PBS and filtered before being purified by using a 1 mL MabSelect SuRe column (Cytiva) according to the manufacturer's protocol. The concentration of the purified 0304-3H3 antibody was quantified using the BCA assay (Thermo Scientific).
8
Secretion and Purification of Tri- and Tetravalent Nanobodies
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Tri-and tetravalent nanobody DNA received in the pSS1 vector contains an N-terminal BCL1 signal sequence that targets the nanobody for secretion from the cells, allowing their extraction from the cell culture media. Mammalian HEK293T cells were cultured at 37 °C and 5% CO 2 in complete Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% penicillin, 1% streptomycin, and 1% glutamine. Tri-and tetravalent nanobody DNA was transiently transfected at 60% cell confluency using polyethylenimine (PEI Max MW 40 000, Polysciences) according to the manufacturer's instructions. Nanobodies were expressed as secreted protein into the culture media, collected after 7 days, and subsequently purified using affinity chromatography using either nickel NTA beads for the trivalent nanobodies or a MabSelect SuRe column (Cytiva) for the Fc-tetravalent nanobodies. Tag cleavage was achieved as described for the divalent nanobodies [13 13. Maqsood, Z. ]. Further purification by size exclusion chromatography on a HiLoad 26/600 Superdex 75 pg column (Cytiva) was used, if necessary, to obtain pure nanobodies. The concentration of purified nanobody was determined using a NanoDrop spectrophotometer (ND-1000, Geneflow) measuring absorbance at 280 nm according to the manufacturer's protocol. Purified nanobodies were stored at -80 °C.
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