Dextrin sepharose high performance

After inoculation of the recombinant baculoviruses P1 for 4 days in the Sf-9 cells, the cellular pellets were collected and resuspended with PBS containing EDTA and cOmplete mini. The cellular lysate was subjected to 2 freeze-thaw cycles and then centrifuged at 18,000 g for 10 min to remove cells and debris. The supernatants were transferred into Dextrin Sepharose High Performance (GE Healthcare) and purification of the MBP-tagged NS1 was performed according to the manufacturer’s protocol. Briefly, samples were poured into a column, washed with the binding buffer [20 mM TrisHCl (pH 7.4), 0.2 M NaCl, 1 mM EDTA and 1 mM DTT] and eluted with the elution buffer [0.5 M maltose, 20 mM TrisHCl (pH 7.4), 0.2 M NaCl, 1 mM EDTA and 1 mM DTT]. The size of the purified NS1 protein was examined by gel filtration using a Superose 6 column (Cytiva). Reaction was carried out at a flow rate of 0.5 ml/min with a buffer [50 mM phosphate buffer (pH 7.4) and 150 mM NaCl]. The loaded samples were fractionated into 39 fractions (500 μl each), and all fractions (20 μl) were analyzed by SDS-PAGE and immunoblotting using anti-NS1 antibody.