Fig. 1
From: Molecular characterization of duck enteritis virus UL41 protein
Purification of the fusion protein and preparation of the polyclonal antibody. All bacteria samples were collected by centrifugation at 4 °C and disrupted by ultrasonication, and then analyzed by SDS-PAGE with 12% resolving gels and 5% stacking gels. a Lane 1, pET-32a(+) vector; Lane 2, recombinant bacterial sediment; Lane 3, recombinant bacterial supernatant; M. Bio-Rad low-molecular-weight protein marker. Protein expression was induced at 37 °C for 6 h with 0.4 mM IPTG until OD600 reached 0.4–0.6. Significantly higher UL41-His recombinant protein levels were observed in the recombinant bacterial sediment. b IPTG concentration optimization. Lanes 1–7, IPTG concentrations of 1.2, 1.0, 0.8, 0.6, 0.4, 0.2, and 0.1 mM, respectively. M. Bio-Rad low-molecular-weight protein marker. Significantly higher UL41-His recombinant protein levels were observed at 0.2 mM. c Induction temperature optimization. Lane 1, induction at 25 °C; Lane 2, induction at 30 °C; Lane 3, induction at 37 °C; M. Bio-Rad low-molecular-weight protein marker. Significantly higher UL41-His recombinant protein levels were observed at 30 °C. d Induction time optimization. Lanes 1–7, induction for 12.5, 10, 8, 6, 4, 2, and 1 h, respectively; M. Bio-Rad low-molecular-weight protein marker. Significantly higher UL41-His recombinant protein levels were observed at 10 h. e Recombinant protein purification. Lane 1, purified protein (approximately 76 kDa); M. Bio-Rad low-molecular-weight protein marker. f Expressed protein identification by western blotting using rabbit anti-DEV antibody (1:800) and goat anti-rabbit IgG HRP-conjugated antibody (1:5000). Lane 1, pET-32a(+) vector; Lane 2, recombinant protein (approximately 76 kDa); M. Precision Plus Protein™ Dual Color Standards