pET31HT-PNC27

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Escherichia coli BL21(DE3) harboring the expression vectors were cultivated in LB medium containing 100 μg/ml ampicillin, at 37°C with shaking. The overnight cultures were used to inoculate 100 ml LB media (with 100 μg/ml ampicillin) to an initial OD600 = 0.05, and grown at 37°C with shaking. When OD600 of the cultures reached 0.6, IPTG was added to a final concentration of 1 mM.

Protocol tips
Escherichia coli BL21(DE3) harboring the expression vectors were cultivated in LB medium containing 100 μg/ml ampicillin, at 37°C with shaking. The overnight cultures were used to inoculate 100 ml LB media (with 100 μg/ml ampicillin) to an initial OD600 = 0.05, and grown at 37°C with shaking. When OD600 of the cultures reached 0.6, IPTG was added to a final concentration of 1 mM.

Publication protocol

Escherichia coli BL21(DE3) harboring the expression vectors were cultivated in LB medium containing 100 μg/ml ampicillin, at 37°C with shaking. The overnight cultures were used to inoculate 100 ml LB media (with 100 μg/ml ampicillin) to an initial OD600 = 0.05, and grown at 37°C with shaking. When OD600 of the cultures reached 0.6, IPTG was added to a final concentration of 1 mM. After 3–6 h induction, cells were harvested by centrifugation, resuspended in binding buffer (40 mM Tris, 500 mM NaCl, 15 mM Imidazole, pH 8.0), and lysed by sonication in an ice-water bath. The suspensions were centrifuged (10,000 x g, 10 min) to pellet the insoluble matter, and the pellets were resuspended in denaturing binding buffer (8 M Urea) and centrifuged again. The supernatants were incubated on a rotary shaker with Ni-NTA agarose at 4°C for 60 min. Then, the columns were packed and washed with denaturing binding buffer and the fused peptides were eluted with elution buffer (40 mM Tris, 500 mM NaCl, 300 mM Imidazole, 8 M Urea, pH 8.0).

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