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  • Protein Expression Using BL21(DE3) (C2527)

    Protocols.io also provides an interactive version of this protocol where you can discover and share optimizations with the research community. There is also an interactive version of this protocol available for the large scale. 

    Protocol

    1. Transform expression plasmid into BL21(DE3). Plate on antibiotic selection plates and incubate overnight at 37°C.
    2. Resuspend a single colony in 10 ml liquid culture with antibiotic.
    3. Incubate at 37°C until OD600 reaches 0.4–0.8.
    4. Induce with 4 or 40 µl of a 100 mM stock of IPTG (final concentration of 40 or 400 µM) and induce for 3 to 5 hours at 37°C.
    5. Check for expression either by Coomassie stained protein gel, Western Blot or activity assay. Check expression in both the total cell extract (soluble + insoluble) and the soluble fraction only.
      *If a fraction of the target protein is insoluble, repeat expression at a lower temperature (15 to 30°C) or test expression in Lemo21(DE3) (NEB #C2528).
    6. For large scale, inoculate 1 L of liquid medium (with antibiotic) with a freshly grown colony or 10 ml of freshly grown culture. Incubate at 37°C until OD600 reaches 0.4–0.8. Add 40 or 400 µM IPTG and express protein using optimal time/temperature determined in a small scale trial.