What is the optimal IPTG concentration and incubation time after induction?
Normally, My protein were induced with 0.5mM at 25'C for O/N but now I have limitation about shaker, so i'll to try new condition at 30-37’C.
Giggsy
If you are talking of protein expression in E.coli, the normally induction for an intracellular protein is 1 mM IPTG at 37°C for 2-4h. As you are expressing a secreted protein it is normal collect the protein accumulated in the medium during a long time of culture, the secretion machinery limit the secretion yield. For short time induction is convenient purify the protein also from the bacterial mass.
Cuture and induction conditions have to be optimized for each protein individually. Unless you use a permease-deficient strain, 0.5 mM IPTG is saturating. Beware that if your protein was produced in soluble form at 25 °C, it may form insoluble incultion bodies if induction is performed at a higher temperature. To summarize, check your new conditions on a small batch before investing in a large culture
HI Giggsy,
I expressed membrane protein in E.Coli, induced with 1 mM IPTG and reduced the temp. to 26 C for 3 hrs (OD600 doubled) then harvested. You may try 0.5 mM or 1mM at 30 C for 3 hrs and see.
Kiran
Please look at this article. May be helpful.
High-level production of membrane proteins in E. coli BL21(DE3) by omitting the inducer IPTG
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