pGEX-4T-1

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The lsrK expression plasmid was transformed into E. coli BL21 (DE3) cells and the antibiotic ampicillin (100 µg ml−1) was used for E. coli to maintain the plasmid. A seed culture with a 1/100th volume of the main culture was prepared by growing the transformed cells in Luria–Bertani (LB) growth medium (Duchefa Biochemie, The Netherlands) overnight at 37°C.	The amount of LsrK expressed was tested with the following three different incubation conditions using a 2.8 l scaled baffled flask containing 1 l LB medium. (i) The cells were grown in LB medium at 37°C until the OD of the culture at 600 nm (OD600) reached 0.7–0.8 and induction of LsrK expression was then performed with 1.0 mM isopropyl β-d-1-thiogalactopyranoside (IPTG; Duchefa Biochemie, The Netherlands) for 4 h.

Protocol tips
The lsrK expression plasmid was transformed into E. coli BL21 (DE3) cells and the antibiotic ampicillin (100 µg ml−1) was used for E. coli to maintain the plasmid. A seed culture with a 1/100th volume of the main culture was prepared by growing the transformed cells in Luria–Bertani (LB) growth medium (Duchefa Biochemie, The Netherlands) overnight at 37°C.
Downstream tips
The amount of LsrK expressed was tested with the following three different incubation conditions using a 2.8 l scaled baffled flask containing 1 l LB medium. (i) The cells were grown in LB medium at 37°C until the OD of the culture at 600 nm (OD600) reached 0.7–0.8 and induction of LsrK expression was then performed with 1.0 mM isopropyl β-d-1-thiogalactopyranoside (IPTG; Duchefa Biochemie, The Netherlands) for 4 h.

Publication protocol

"The lsrK expression plasmid was transformed into E. coli BL21 (DE3) cells and the antibiotic ampicillin (100 µg ml−1) was used for E. coli to maintain the plasmid. A seed culture with a 1/100th volume of the main culture was prepared by growing the transformed cells in Luria–Bertani (LB) growth medium (Duchefa Biochemie, The Netherlands) overnight at 37°C. The amount of LsrK expressed was tested with the following three different incubation conditions using a 2.8 l scaled baffled flask containing 1 l LB medium. (i) The cells were grown in LB medium at 37°C until the OD of the culture at 600 nm (OD600) reached 0.7–0.8 and induction of LsrK expression was then performed with 1.0 mM isopropyl β-d-1-thiogalactopyranoside (IPTG; Duchefa Biochemie, The Netherlands) for 4 h. (ii) The cells were grown in LB medium containing 0.4% glucose at 37°C until the OD600 reached 0.5–0.6 and the temperature of the incubator was then changed to 20°C. When the temperature reached 20°C, IPTG induction was performed for 20 h at the same temperature. (iii) For the expression of LsrK in the presence of osmotic shock and heat shock, the cells were grown in LB medium containing an additional 250 mM NaCl and 0.4% glucose at 37°C. When the culture OD600 reached 0.5–0.6, the culture flasks were immediately transferred to a different shaking incubator with the temperature preset to 42°C. After 1 h, the cells were cooled on ice for 30 min and IPTG induction was then performed at 20°C for an additional 20 h. The cells were then harvested by centrifugation at 2500g and 4°C for 30 min.

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Check out relevant papers found by Labettor's AI that are relevant for performing Protein Expression Prokaryotic cells - E. coli LsrK using pGEX-4T-1 from Kyoung-Seok Ryu, Protein Structure Group, Korea Basic Science In.

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Download the product protocol from Kyoung-Seok Ryu, Protein Structure Group, Korea Basic Science In for pGEX-4T-1 below.

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