pcDNA-oGP1

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	For transient expression, a T-25 flask was seeded with 1 ×106 293T cells one day before the transfection. Six μg of pcDNA-nGP1 or pcDNA-oGP1 was diluted into 0.3 ml OPTI-MEM I reduced serum medium and 24 μl of FuGENE® HD Transfection Reagent (Promega, Madison, WI) was then added into the solution. The transfection reagent and DNA mix was incubated for 20 min at room temperature, then added into the T-25 flask.

Protocol tips

For transient expression, a T-25 flask was seeded with 1 ×106 293T cells one day before the transfection. Six μg of pcDNA-nGP1 or pcDNA-oGP1 was diluted into 0.3 ml OPTI-MEM I reduced serum medium and 24 μl of FuGENE® HD Transfection Reagent (Promega, Madison, WI) was then added into the solution. The transfection reagent and DNA mix was incubated for 20 min at room temperature, then added into the T-25 flask.

Publication protocol

293T or 293-H cells (Invitrogen) were grown in DMEM/F12 medium containing 5% FBS. For transient expression, a T-25 flask was seeded with 1 ×106 293T cells one day before the transfection. Six μg of pcDNA-nGP1 or pcDNA-oGP1 was diluted into 0.3 ml OPTI-MEM I reduced serum medium and 24 μl of FuGENE® HD Transfection Reagent (Promega, Madison, WI) was then added into the solution. The transfection reagent and DNA mix was incubated for 20 min at room temperature, then added into the T-25 flask. The medium was replaced after 24 h and cells were cultured continuously for another 5 days. Supernatants were then taken for an ELISA expression test. To establish a stable GP1 expressing cell line, 293-H cells were transfected in the same way with pcDNA-oGP1 plasmid, then seeded into 384-well plates and cultured with DMEM/F12 medium containing 5% FBS and 500 μg/ml G418 two days post transfection. After approximately two weeks, colonies were screened for their GP1 expression by ELISA. Briefly, 1 μg of mAb6D8 antibody was immobilized onto each well of an ELISA plate. Horseradish peroxidase labeled rabbit anti-6X His tag polyclonal antibody (Abcam, Cambridge, MA) was used as a detection antibody with a dilution of 1/5000. The clone with the highest expression level of GP1 was chosen for recombinant protein production.

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