All proteins were expressed and purified from Expi293 BirA cells according to established protocol from the manufacturer (Thermo Fisher Scientific). Briefly, 30 μg of pFUSE (InvivoGen) vector encoding the protein of interest was transiently transfected into 75 million Expi293 BirA cells using the Expifectamine kit (Thermo Fischer Scientific). For the IgG and Fab proteins, 15 μg of each chain was transfected. Enhancer was added 20 h after transfection. Cells were incubated for a total of 3 d at 37 °C in an 8% CO2 environment before the supernatants were harvested by centrifugation. Fc-fusion proteins were purified by Protein A affinity chromatography and His-tagged proteins were purified by Ni-NTA affinity chromatography. Purity and integrity were assessed by SDS/PAGE. Purified protein was buffer exchanged into PBS and stored at −80 °C in aliquots.
Expifectamine kit
Manufactured by Thermo Fisher Scientific
The Expifectamine kit is a laboratory reagent designed for the transfection of mammalian cells. It facilitates the introduction of nucleic acids, such as plasmid DNA or mRNA, into the cells. The kit provides a ready-to-use solution for efficient transfection, allowing researchers to study gene expression, protein production, and other cellular processes.
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Lab products found in correlation
3 protocols using expifectamine kit
1
Transient Protein Expression in Expi293 Cells
2
Recombinant Protein Expression and Purification
3
Monoclonal Antibody Production and Purification
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Monoclonal antibodies PGT121, PG9, VRC01, and 10E8 were produced with plasmids obtained through the NIH HIV Reagent Program, Division of AIDS, NIAID, NIH. Heavy and light chain plasmids were transfected into 293i cells using the Expifectamine kit (ThermoFisher A14524) as per manufacturer guidelines. Transfected cells were incubated for 5 days before harvesting the cell supernatant. Antibodies were purified from culture supernatants using affinity chromatography with protein A beads as previously described (47) . After purification, all antibodies were then buffer exchanged into a citric acid buffer (pH 6) before use.
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