pFastBac-N

Protein Expression Eukaryotic cells - S. frugiperda AvCoV-IBV

Experiment
Protein Expression Eukaryotic cells - S. frugiperda AvCoV-IBV
Product
pFastBac-N from Juergen A. Richt, Department of Diagnostic Medicine and Pathobio
Manufacturer
Juergen A. Richt, Department of Diagnostic Medicine and Pathobio

Protocol tips

Protocol tips
recombinant bacmids were purified using HighPure MiniPrep Kit (Life Technologies) and used to transfect Spodoptera frugiperda (Sf9) cells grown in Sf-900 II Serum-free Medium (SFM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin as manufacturer’s instruction (Life Technologies). Transfection was carried out using Cellfectin II reagent as previously described [8] and according to the manufacturer’s instructions (Invitrogen-Life Technologies).

Publication protocol

To rescue recombinant baculoviruses encoding the IBV N gene, recombinant bacmids were purified using HighPure MiniPrep Kit (Life Technologies) and used to transfect Spodoptera frugiperda (Sf9) cells grown in Sf-900 II Serum-free Medium (SFM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin as manufacturer’s instruction (Life Technologies). Transfection was carried out using Cellfectin II reagent as previously described [8] and according to the manufacturer’s instructions (Invitrogen-Life Technologies). Recombinant IBV N protein was expressed using passage 2 or higher passage recombinant baculovirus stocks (> 107 pfu/ml). The protein was expressed with a carboxy-terminal 6xHis tag, and purification using Ni-NTA Superflow resin (QIAGEN Inc., Valencia, CA) was performed as described previously [7]. Concentration of the purified protein was measured by the method of bicinchoninic acid (BCA) assay (Thermo Scientific, Rockford, IL) at an absorbance of 562 nm, using bovine serum albumin (Sigma-Aldrich, St. Louis, MO) as the protein standard. Aliquots of the protein were stored at − 80 C until used.

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