Protein Expression Using BL21(DE3) (C2527)

Abstract

This protocol explains methods for Protein Expression Using BL21(DE3) Competent E. coli (C2527).

You can find the protocol for a large-scale Protein Expression here.

Before start

Steps

Transform expression plasmid into BL21(DE3).

Plate on antibiotic selection plates.

Incubate at 37°C.

Resuspend a single colony in 10mL.

Incubate at 37°C until OD₆₀₀ reaches 0.4–0.8.

Induce with 4µL or 40µL of 100millimolar (mM) (final concentration of 40 or 400 µM).

Induce for 3-5 hours at 37°C.

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.

Note

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) (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).).

For large scale, inoculate 1L of liquid medium (with antibiotic) with a freshly grown colony or 10mL of freshly grown culture.

10.

Incubate at 37°C until OD₆₀₀reaches 0.4–0.8.

11.

Add 40micromolar (µM) or 400micromolar (µM) IPTG and express protein using optimal time/temperature determined in a small scale trial.

Abstract

Before start

Steps

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