Previously published plasmablast antibody sequences from RA patients (48 (link), 69 , 70 (link)) were recombinantly produced in human IgG1 backbone to ensure consistency in the characterization assays. In-house production was done as described previously, using an Expi293 Expression System (ThermoFisher) with Expi293F cells (48 (link), 69 –71 (link)). Briefly, constructs including the heavy chain and light chain variable region sequences were synthesized as gBlock gene fragments (IDT) for cloning into pFUSE antibody plasmids (Invivogen) using the Cold Fusion Cloning Kit (System Biosciences). We utilized pFUSEss-CHIghIg1 for gamma, pFUSE2ss-CLIg-hK for kappa, and pFUSE2ss-CLIg-hL2 for lambda. The Expi293 Expression System (ThermoFisher Scientific) was used for transient transfections, and harvested culture supernatants were purified using Pierce™ Protein A Plus Agarose (ThermoFisher Scientific).
Pierce protein a plus agarose
Manufactured by Thermo Fisher Scientific
Pierce™ Protein A Plus Agarose is a high-capacity agarose resin designed for the purification of antibodies and immunoglobulins. It provides a stable, cross-linked agarose matrix with covalently coupled recombinant Protein A ligand.
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4 protocols using pierce protein a plus agarose
1
Recombinant Plasmablast Antibody Production
2
IgG Purification from BY-2 Culture
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Eight × 50 mL of a 7-day old BY-2 suspension culture was filtered on three layers of Miracloth (Calbiochem) and the filtrate was centrifuged (8,000 g, 30 min). The supernatant was recovered, supplemented with 10% 1 M Tris-Cl pH 8.0 and incubated for 16 h at 4°C with one mL of Pierce® Protein A Plus Agarose (Thermoscientific # 22812) previously washed three times with 10 mL of 0.1 M Tris-Cl pH 8.0. The sample was then filtered through a nylon membrane and poured onto a poly-prep® chromatography column (Biorad # 731-1550). After washing with 10 mL of 0.1 M Tris-Cl pH 8.0, IgG were eluted with 8 × 500 μL 0.1 M glycin pH 3.0 and immediately supplemented with 10% 1 M Tris-Cl pH 8.0. Ten microlliter of each elution fraction were used for SDS-PAGE (8% acrylamide) analysis followed by colloidal blue staining.
3
Co-immunoprecipitation of SARS-CoV-2 nsp12
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Immunoprecipitation assays were performed as previously described [15 (link)]. Briefly, 293T cells were transfected with a FLAG-tagged nsp12-expressing plasmid (pCMV-FLAG-CoV-2-nsp12). After 72 h of incubation, 1 × 107 cells were lysed in 1 ml of Lysis buffer A (0.5% NP-40, 20 mM Tris–HCl (pH 7.4), and 150 mM NaCl). After sonication, lysates were cleared of insoluble material by centrifugation at 21,000×g at 4 °C for 15 min. FLAG-nsp12 proteins were co-immunoprecipitated using Pierce Protein A Plus Agarose (Thermo Fisher Scientific) and 20 µg of anti-nsp12 mAb (RdMab-2) from the lysate, and then precipitated immune complexes were eluted in 2 × SDS loading buffer (2% β-mercaptoethanol, 20% glycerol, 4% SDS, and 100 mM Tris–HCl (pH 6.8)). The eluted proteins were detected by western blotting analysis. To detect the precipitated FLAG-nsp12 proteins, anti-FLAG mouse mAb (1:5000; M2) and Mouse TrueBlot ULTRA Anti-Mouse Ig HRP (1:4000; Rockland, Gilbertsville, PA) were used for western blotting analysis.
4
Immunoprecipitation Assay of c-JUN in MDA-MB-231 Cells
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For immunoprecipitation assay, MDA-MB-231 cells were transfected with an expression plasmid encoding c-JUN (pcDNA3.1/V5-His-c-JUN) or GFP as a control (pMAX/GFP). After 48 h, the cells were collected, and total proteins were lysed in a lysis buffer (50 mM Tris-HCl, pH 7.9, 10 mM KCl, 400 mM NaCl, 1 mM EDTA, 0.2 % NP-40, 10 % glycerol, 1 mM phenylmethylsul-phonyl fluoride and a cocktail of protease inhibitors). The cell lysates were incubated with the anti-V5 antibody (Cell Signaling Technology) overnight at 4 °C. Antibody-bound protein complexes were precipitated using Pierce Protein A Plus agarose (Thermo Fisher Scientific). Agarose beads were washed thrice with the lysis buffer and heated in a sodium dodecyl sulphate (SDS) sample loading buffer at 95 °C for 5 min. The eluted samples were separated by SDS-poly acrylamide gel electrophoresis, and immunoblotting analysis was performed using anti-V5 and anti-HDAC1 antibodies. Blots were visualized using an enhanced chemiluminescence detection system (Amersham Phar macia Biotech).
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