VH and VL of the positive phage were subcloned, respectively, into the pFUSEss-CHIg-hG1 and pFUSEss-CLIg-hK (Invivogen). Antibodies were transiently expressed in FreeStyle™ HEK 293-F cells (Life Technologies) using 293fectin transfection reagent according to manufacturer’s instructions. After transfection, cells were grown in the serum-free medium for an additional 5 days. The supernatant was collected and purified on a MabSelect Protein A column (GE healthcare). Eluted IgG was dialyzed against PBS and stored at −80°C.
Mabselect protein a column
Manufactured by GE Healthcare
Sourced in Sweden
The MabSelect Protein A column is a chromatography resin designed for the purification of monoclonal antibodies. It features a Protein A ligand immobilized on a highly cross-linked agarose matrix, which enables the capture and separation of antibodies from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Freestyle hek 293 f cells, by Thermo Fisher Scientific (1 mentions)
Pfusess clig hk, by InvivoGen (1 mentions)
Pfusess chig hg1, by InvivoGen (1 mentions)
Human il 2, by Thermo Fisher Scientific (1 mentions)
Superdex s 200 size exclusion column, by GE Healthcare (1 mentions)
Rituximab, by Roche (1 mentions)
2 protocols using mabselect protein a column
1
Recombinant Antibody Production in HEK Cells
2
Affinity Purification and Characterization of ICKs and Antibodies
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The ICKs and antibodies were affinity-purified from culture supernatants by using a MabSelect Protein A column (GE Healthcare, Sweden) and size exclusion/exchange chromatography. ICK and antibodies concentration was determined by measuring the absorbance at 280nm with a molar extinction coefficient of 1.431 and purity was analyzed in non-reducing conditions on SDS-PAGE, and on an analytical Superdex S-200 size exclusion column (GE Healthcare, Sweden).
Rituximab, a chimeric mAb that recognizes human CD20 molecule, was purchased from Roche. MOPC-173 mAb (Abcam) was used as isotype-matched control. The no-alpha mutein was produced and purified at CIM as previously described (25 (link)). The human IL-2 was purchased from Peprotech. Of note, human IL-2 (hIL-2) binds to mouse βγ and αβγ IL-2Rs (12 (link)).
Rituximab, a chimeric mAb that recognizes human CD20 molecule, was purchased from Roche. MOPC-173 mAb (Abcam) was used as isotype-matched control. The no-alpha mutein was produced and purified at CIM as previously described (25 (link)). The human IL-2 was purchased from Peprotech. Of note, human IL-2 (hIL-2) binds to mouse βγ and αβγ IL-2Rs (12 (link)).
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