Codon-optimized cDNA for full-length human Nav1.7 (Uniprot Q15858), a gift from Tsinghua University, was cloned into the pCAG vector with Twin-Strep-tag and Flag-tag in tandem at the amino terminus while codon-optimized cDNAs for β1 subunit (Uniprot Q07699) and β2 subunit (Uniprot O60939) were cloned separately into the pCAG vector without affinity tag. All the plasmids for transient expression were verified by DNA sequencing. HEK293F suspension cells (Thermo Fisher Scientific, R79007) were cultured in SMM 293T-II medium (Sino Biological Inc.) at 37 °C, supplied with 5% CO2 under 60% humidity, and transfected with plasmids when the cell density reached 1.5–2.0 × 106 cells per ml. For one-liter cell culture, a mixture of expression plasmids including 1.5 mg plasmids for Nav1.7, 0.5 mg plasmids for β1, and 0.5 mg plasmids for β2 was pre-incubated with 4 mg 40-kDa linear polyethylenimines (Polysciences) in 50 ml fresh medium for 15–30 minutes, and then added to the cell culture for transient expression of human Nav1.7 complex.
40 kda linear polyethylenimines
Manufactured by Polysciences
40-kDa linear polyethylenimines are high molecular weight cationic polymers commonly used in various laboratory applications. These polymers are water-soluble and can be utilized for diverse purposes, such as transfection reagents, polymer coatings, and other research-oriented functions.
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2 protocols using 40 kda linear polyethylenimines
1
Transient Expression of Human Nav1.7 Complex
2
Transient Expression of Human Nav1.6 Complex
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Codon-optimized cDNA for full-length human Nav1.6 (Uniprot Q9UQD0), a gift from Tsinghua University, was cloned into the pCAG vector with amino-terminal Twin-Strep-tag and FLAG-tag in tandem, while codon-optimized cDNAs for human Nav β1 subunit (Uniprot Q07699) and human FHF2B (Uniprot Q92913-2) were cloned separately into the pCAG vector without any affinity tag. All the plasmids for transient expression were verified by PCR sequencing. Transient expression of Nav1.6-β1-FHF2B adopted our established protocol with slight modifications (28 (link)). Briefly, HEK293F suspension cells (Thermo Fisher Scientific, R79007) were cultured at 37 °C in SMM 293T-II medium (Sino Biological Inc.). A plasmid mixture of 1.5 mg pCAG-Nav1.6, 0.5 mg pCAG-β1 and 0.5 mg pCAG-FHF2B was pre-incubated with 4 mg 40-kDa linear polyethylenimines (Polysciences), then added into 1 L of cell culture with a cell density of 1.5 to 2.0 × 106 cells per mL for transient expression. Transfected cells were harvested approximately 48 h after transfection.
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