pET32a (+)-Glu-Em7

Protein Expression Prokaryotic cells - E. coli B. subtilis Em7

Experiment
Protein Expression Prokaryotic cells - E. coli B. subtilis Em7
Product
pET32a (+)-Glu-Em7 from L.L. Huang, State Key Laboratory of Crop Stress Biology for Arid
Manufacturer
L.L. Huang, State Key Laboratory of Crop Stress Biology for Arid

Protocol tips

Protocol tips
The recombinant strain E. coli BL21 (DE3)/pET32a (+)-Glu was inoculated in 5 mL LB liquid medium (containing 100 μg/mL ampicillin) and cultured at 37°C overnight. Then, 1 mL of this culture was inoculated into 100 mL LB medium (containing 100 μg/mL ampicillin) and grown at 37°C with shaking at 200 rpm until the OD600 value reached 0.4-0.6.

Publication protocol

The recombinant strain E. coli BL21 (DE3)/pET32a (+)-Glu was inoculated in 5 mL LB liquid medium (containing 100 μg/mL ampicillin) and cultured at 37°C overnight. Then, 1 mL of this culture was inoculated into 100 mL LB medium (containing 100 μg/mL ampicillin) and grown at 37°C with shaking at 200 rpm until the OD600 value reached 0.4-0.6. At this time, IPTG was added at a final concentration of 0.5, 1, 2, 4, 6, or 8 mM, and the culture was incubated for another 6 h at 30°C, shaking at 200 rpm. For the controls, E. coli BL21 (DE3)/pET32a strain induced with 1 mM IPTG and E. coli BL21 (DE3)/pET32a Glu without IPTG induction were used. The cell pellet from 1-mL culture, obtained by centrifugation at 10,621 g for 5 min, was resuspended in 100 μL 20 mM PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2 HPO4, and 1.8 mM KH2PO4, pH 7.3) and 8μL lysozyme (50 mg/L) at 37°C for 1 h. The mixture was then subjected to 4-5 freeze-thaw cycles using liquid nitrogen and the supernatant was obtained by centrifugation at 15,294 g for 10 min. Finally, 15 μL of this supernatant was analyzed by sodium dodecyl sulfate (SDS)-PAGE using the Mini-protean III system (Bio-Rad, Beijing, China). Protein bands were visualized by Coomassie brilliant blue R-250 staining.

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Manufacturer protocol

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