Profinity imac uncharged column

Recombinant proteins were produced and purified using Drosophila Expression System (DES^®, Life technologies). Briefly, pMT-BIP-CD83 scFv-V5-His, pMT-BIP-rH9HA-V5-His, or pMT-BIP-rH9HA-CD83 scFv-V5-His plasmids were co-transfected into Drosophila melanogaster S2 cells together with a hygromycin B resistance plasmid (pCoHYGRO, Life technologies). Antibiotic selection was carried out for four weeks using hygromycin B at a concentration of 250 µg/ml and a single cell clone was obtained via limiting dilution^{43 (link)}. Recombinant proteins were secreted into culture supernatant after CuSO₄ (500 µM) induction and then purified by Profinity™IMAC uncharged column (Bio-Rad). Protein expression was determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using 10−12% gradient polyacrylamide gels (Bio-Rad) followed by Coomassie staining (Life Technologies). Furthermore, proteins were also transferred onto nitrocellulose membranes (Amersham) for western blotting using anti-H9HA monoclonal antibodies^{44 (link)}. The concentration of purified recombinant proteins was determined by Pierce™ BCA Protein Assay Kit (Life Technologies) according to the manufacturer’s protocol.

All the recombinant proteins were expressed using Drosophila Expression System (DES^®, Life technologies). The expression plasmids were transfected into Drosophilla melanogaster Schneider 2 (S2) cells using calcium phosphate transfection kit, according to manufacturer’s protocol (Life Technologies). Stable S2 transfectants were generated by adding hygromycin to a final concentration of 250 μg/mL every week for at least 4 weeks. Single cell clone expressing recombinant proteins was obtained via the limiting dilution method [34 (link)]. Recombinant proteins were secreted into culture supernatant after CuSO₄ (500 µM) induction and then purified using Profinity™ IMAC uncharged column (Bio-Rad, Hercules, CA, USA). The purified proteins were analysed by using 10–12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie blue (Life Technologies) staining. The concentration of the purified proteins was measured using Pierce BCA Protein Assay Kit (Life Technologies) according to the manufacturer’s protocol.