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Test Induction of Protein Expression in E.coli

 

The night before expression:

Inoculate 5mL 2XYT culture with one colony (or scraping of glycerol stock) of your construct of interest transformed into BL21 E.coli.  Don’t forget to add antibiotic1:1000.  Shake cultures o/n at 37°C overnight.

 

In the morning:

Inoculate 5-10mL of 2XYT with 100-200 uL of your overnight culture.  Remember your antibiotic. 

 

Shake cultures at 37°C for approximately 2 hours, then check OD600 of culture.

 

Grow culture to an OD600 of 0.5- 0.9 (I usually shoot for an OD of 0.8).  Take a 0.5mL sample of culture: this is your “uninduced” sample.  Pellet the bacteria at max speed in the minicentrifuge, remove the supernatant and resusupend pellet in 100mL of 2X SDS-PAGE sample buffer.  Freeze at -20°C until ready for gel.

 

For expression of Xenopus proteins, cool cultures in an icebath to 20°C using a thermometer cleaned with ethanol to monitor temperature.

 

Induce protein expression with IPTG (final concentration 1mM for pET vectors, 0.4mM for pGEX vectors). 

 

Shake cultures at 20°C for 3-5 hours.  Take a 0.5 mL sample of induced culture at appropriate times.  These are “induced” samples.  Pellet as before, and add sample buffer. 

 

Sonicate each sample to break up DNA.  Boil samples for 5 minutes in the heating block on the weigh bench.  Load 5 uL of each sample on an SDS-PAGE gel, loading uninduced samples next to the appropriate induced sample(s).

 

 

This page last modified 08/11/2011

The Stukenberg Lab and the Burke Lab are in the Department of Biochemistry and Molecular Genetics at the University of Virginia