Heavy and light chain amino acid sequences were downloaded from the CoV-AbDab database and synthesized as codon-optimized gBlock fragments (GENEWIZ). These antibodies were expressed using the Expi293 expression system kit (Cat# A14635, Thermo Fisher) with human IgG1 constant regions. The culture supernatant was collected and loaded at 4 mL/min on a 5 mL HiTrap Protein G column (Cytiva, Marlborough, MA, USA) equilibrated with 10 mM phosphate, pH 7, using a Bio-Rad NGC fast protein liquid chromatography (FPLC) system. The medium was tittered to pH 7 with 1 M monosodium phosphate before loading. The antibodies were eluted with 100 mM glycine, pH 2.7, and collected in tubes containing 1 M dibasic sodium phosphate to neutralize the pH. The eluate was concentrated to <4 mL with a 4 mL 50 kDa molecular weight cut-off (MWCO) Amicon ultracentrifuge filter, and the buffer was exchanged on a 10 mL Zeba desalting column (ThermoFisher) equilibrated in PBS. The final antibody concentration was determined using the protein extinction coefficient for IgG ( ).
Hitrap protein g column
Manufactured by Cytiva
Sourced in United States
The HiTrap Protein G column is a ready-to-use affinity chromatography column designed for the purification of antibodies. It contains Protein G, a bacterial protein that binds to the Fc region of immunoglobulins, immobilized on agarose beads. The column can be used for the rapid and efficient capture and purification of antibodies from complex samples, such as cell culture supernatants or sera.
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Lab products found in correlation
Hitrap, by Cytiva (10 mentions)
Ez link sulfo nhs biotinylation kit, by Thermo Fisher Scientific (2 mentions)
Polyethylene glycol, by Merck Group (2 mentions)
Zeba desalting column, by Thermo Fisher Scientific (1 mentions)
Expi293 expression system kit, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Escherichia coli nico21 de3 cells, by New England Biolabs (1 mentions)
Complete freund s adjuvant, by Merck Group (1 mentions)
Incomplete freund s adjuvant, by Merck Group (1 mentions)
Expi293 expression system, by Thermo Fisher Scientific (1 mentions)
Kta pure system, by Cytiva (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Pierce fab preparation kit, by Thermo Fisher Scientific (1 mentions)
Hygromycin b, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
10 protocols using hitrap protein g column
1
Recombinant Antibody Production and Purification
2
SARS-CoV-2 Spike Glycoprotein Detection
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After SDS-PAGE proteins were transferred onto nitrocellulose membranes (BioRad, USA) at 90 V for 1 h. Membranes were then incubated with 30 ml Tris-buffered saline with Tween (TTBS) buffer (pH 8.0) containing 5% nonfat dry milk for 1 h at 20°C. Membranes were stained with a 1:1000 dilution of COVID-19 convalescent serum or rabbit anti-protein S serum (rabbits were immunized twice with 150 µg of recombinant human coronavirus SARS-CoV-2 Spike Glycoprotein (ab273068, Abcam, UK) with three weeks interval and immunoglobulins were purified on HiTrap protein G column (Cytiva) by incubating overnight at 4°C). After washing with TTBS buffer, the membranes were incubated with a secondary antibody (anti-Human or anti-Rabbit IgG (H + L), HRP conjugate, Promega, USA), diluted 1:1000 for 1 h. The membranes were finally washed with TTBS before the Clarity Western substrate (BioRad) was applied, and visualized on films.
3
Purification and Antibody Generation of ORF29 Nuclease
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The nuclease domain of ORF29 (residues 442–687) containing an N-terminal 6xHis tag and TEV site was produced in Escherichia coli NiCo21 (DE3) cells (New England Biolabs) as previously described in https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6069193/ . Purified ORF29b was used for immunization of two rabbits by YenZym Antibodies LLC (Brisbane, CA). Sera was purified in-house over a HiTrap Protein G column (Cytiva Life Sciences) followed by positive and negative selection with ORF29b-AminoLink and MBP-AminoLink columns generated using the AminoLink Plus Coupling Resin kit (Thermo Scientific) according to the manufacturer’s instructions. The final ORF29b antibody was concentrated to 1 mg/mL and used at a final concentration of 1 μg/mL.
4
Monoclonal Antibody Generation against Tau Protein
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We generated 19 new monoclonal antibodies (mAbs) against tau by immunizing 8‐week‐old Balb/c mice with a recombinant tau241‐441 protein (100 μg) in complete Freund's adjuvant (Sigma). After two to three additional dosages of the recombinant protein (100 μg/mouse) in Freund's Incomplete adjuvant (Sigma), mice were sacrificed, the spleen removed, and B‐cells fused with the myeloma cell line SP2/0 following standard procedures. Approximately 10 days after the fusion, the cell media were screened for tau antibodies using full‐length recombinant tau1–441 (2N4R) as well as the tau241–441 construct by direct enzyme‐linked immunosorbent assay (ELISA). Clones that reacted positively with the recombinant tau proteins were further grown, subcloned, and subsequently frozen in liquid nitrogen. Antibody specificity against tau was verified, and the isotype was determined using a commercially available kit (Pierce Rapid Isotyping Kit‐Mouse). Finally, antibodies were purified using a Hitrap protein G column (Cytiva) according to the manufacturer's instructions. Epitope mapping for each mAb was performed by direct ELISA against five recombinant overlapping (10 amino acids) peptides spanning the tau241–441 sequence (Caslo ApS, Denmark), specifically tau241–291, tau281–331, tau321–371, tau361–411, and tau 401–441.
5
Purification of Anti-HCV Monoclonal Antibodies
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The HCV anti-E2 human monoclonal antibodies (HmAb) CBH-4B, CBH-7, HC-1, HC-11 and HC33.1 have been described previously [20 (link),21 (link)] and were a generous gift from Steven Foung. The sequences of the heavy and light chain sequences of monoclonal antibody 1:7 were obtained from a publish patent, synthesized and subcloned into the appropriate pFuse-ss human IgG vectors (Invivogen, San Diego, USA) [22 (link)]. The plasmids were verified by Sanger sequencing then expressed in the Expi293 expression system as per the manufacturers’ instructions (ThermoFisher Scientific, Waltham, MA, USA). After 3–5 days the medium was harvested, and IgG was purified using a HiTrap Protein G column on an ÄKTA Pure system (Cytiva, Marlborough, MA, USA).
6
Generating Human Chimeric B2 Antibodies
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To produce B2 mAbs in large quantity for in vivo studies, we cloned the variable regions of B2 from hybridoma cells and grafted them onto human IgG1 constant region. The chimeric antibody was expressed in Chinese hamster ovary (CHO) cells. The pcDNA5_FRT plasmid purchased from Invitrogen was modified to insert the variable regions of B2 heavy and light chains in human IgG1 constant regions. Then the plasmid was transfected into CHO_Flp cells using lipofectamine 3000 Reagent (Invitrogen), and the positive clones were screened out over 10 days in the presence of Hygromycin B (50 mg/ml, Gibco). Cells with positive expression of the antibody were grown in CD CHO AGT expression medium (Thermo Fisher Scientific) with supplemental glutamine, D+ glucose, and Pen/Strep for 2 weeks. The media was then harvested, and the secreted antibody (IHH-IgG) was purified using a 5-ml HiTrap protein G column (Cytiva). The antibodies were buffer exchanged into PBS, and the concentration was determined using NanoDrop Spectrophotometer (Thermo Fisher Scientific). The purity of each batch was examined using SDS-PAGE. Purified antibodies were stored at 4°C before experiments. For B2-Fab, human chimeric B2 were digested with immobilized papain using Pierce Fab Preparation Kit (Thermo Fisher Scientific) following the instructions.
7
IgG Fractionation via Protein G
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Pre-immune and terminal bleed sera (1 mL each) were dialyzed into sodium phosphate buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.0) and loaded on a 1 mL HiTrap Protein G column (Cytiva, Vancouver, Canada) connected to an ÄKTA FPLC protein purification system (Cytiva). The G1 (IgG2b) fraction was eluted with 100 mM citrate buffer, pH 3.5, and the G2 (IgG1) fraction was eluted with 100 mM glycine buffer, pH 2.7. Both the G1 and G2 fractions were immediately dialyzed into sodium phosphate buffer.
8
Purifying IgG from HCV+ Patients
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Blood samples were collected from HCV + patients under ethical approval: “Characterising and modifying immune responses in chronic viral hepatitis”; IRAS Number 43993; REC number 11/LO/0421. Extracted serum was heat inactivated, filtered and diluted 1:1 in PBS. Total IgG was captured using a HiTrap protein G column (Cytiva, MA, USA), eluted in pH2.7 glycine buffer and buffer exchanged into PBS. For experiments, batches of pooled IgG were created by equimolar combination of IgG from two patient samples.
9
Purification and Characterization of IgM
10
Purification and Characterization of IgM
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IgM antibody was purified from wild-type mouse serum. Large-scale purification was performed using a HiTrap Protein G column to remove IgG (Amersham Biosciences, Piscataway, NJ). Proteins were precipitated with polyethylene glycol (Sigma) and the product was run over a size exclusion column to isolate the IgM fractions. The purity of the isolated IgM was established by Coomassie staining and Western blot analysis. The IgM was biotinylated using a EZ-link Sulfo-NHS Biotinylation Kit (ThermoScientific) according to the manufacturer's instructions, and binding of the biotinylated IgM to mesangial cells was confirmed by flow cytometry. Monoclonal natural IgM hybridomas were generated from the B cells of C57BL/6 mice as previously described.34 (link) Mice were injected with 1 mg of purified polyclonal IgM or 100 μg of the IgM monoclonal antibodies intravenously by tail vein (N=3 for each antibody preparation). For mice injected with polyclonal IgM, urine and serum samples were collected at baseline and on days 1, 2, and 3 following injection. Kidney tissue was obtained on days 1 and 3. Mice injected with the IgM monoclonal antibodies were sacrificed after 24 hours and the kidney tissue was analyzed.
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