pET22β

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Recombinant cells were cultured in LB/Amp media and were incubated at 37 °C to OD600 = 0.6–0.7. To induce protein expression, 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added and growth was continued for 18 h; in additional trials, different concentrations of IPTG (0.5 mM, 1 mM and 1.5 mM) were used. Effects of a temperature gradient (25 °C, 31 °C and 37 °C) and time variation (3, 6 and 18 h) were considered.	The expressed protein analysis was performed using sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE).

Protocol tips
Recombinant cells were cultured in LB/Amp media and were incubated at 37 °C to OD600 = 0.6–0.7. To induce protein expression, 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added and growth was continued for 18 h; in additional trials, different concentrations of IPTG (0.5 mM, 1 mM and 1.5 mM) were used. Effects of a temperature gradient (25 °C, 31 °C and 37 °C) and time variation (3, 6 and 18 h) were considered.
Downstream tips
The expressed protein analysis was performed using sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE).

Publication protocol

Recombinant cells were cultured in LB/Amp media and were incubated at 37 °C to OD600 = 0.6–0.7. To induce protein expression, 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added and growth was continued for 18 h; in additional trials, different concentrations of IPTG (0.5 mM, 1 mM and 1.5 mM) were used. Effects of a temperature gradient (25 °C, 31 °C and 37 °C) and time variation (3, 6 and 18 h) were considered. The expressed protein analysis was performed using sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS-PAGE). Negative controls including of the E. coli/BL21/pET22 and E. coli/RosettaTM/pET22 were used for each analysis. The recombinant beta protein, which contains a 6-His tag at the C-terminal, was purified with Ni-NTA resin. The bacterial pellet was suspended in the lysis buffer and the cells were lysed with sonication repeated six times on ice for a duration of 5 min. The cell lysate was centrifuged at 13 680 g, and the clarified supernatant was loaded on Ni-NTA resin at the flow rate of 1 mL/min. The column was washed with five volumes of wash buffer, and finally, the protein was eluted by adding elution buffer, as previously described (Pilehchian Langroudi et al. 2013). The purified protein was analysed using SDS-PAGE and western blot. The protein concentration was determined using a standard procedure, as previously described (Bradford 1976

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