pcDNA-nGP1

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.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Protein Expression Eukaryotic cells - HEK293 GP1 using pcDNA-nGP1 from Peter D. Sun, Structural Immunology Section, Laboratory of Immun.

Manufacturer protocol

Download the product protocol from Peter D. Sun, Structural Immunology Section, Laboratory of Immun for pcDNA-nGP1 below.

Videos

Check out videos that might be relevant for performing Protein Expression Eukaryotic cells - HEK293 GP1 using pcDNA-nGP1 from Peter D. Sun, Structural Immunology Section, Laboratory of Immun. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.