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Protein Expression Using BL21(DE3) (C2527)

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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.