From 2007.igem.org

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• Applications:
• Inducing bacteria to produce specific proteins

• Time to complete protocol:
  • Lab time: 15min, 5min, 5min
  • Waiting time:2h, 1h, 30min
• Approximate cost of materials: $

Contents 1 Method from primary and secondary reagents 1.1 Primary & secondary Reagents Required including controls 1.2 Method including controls 1.3 Equipement Required 1.4 References

Method from primary and secondary reagents

Primary & secondary Reagents Required including controls

• LB
• Selective Antibiotic
• Bacterial Colony containing inducible protein plasmid
• IPTG
• PBS
• Triton-X 1000 (or alternative non-ionic detergent)
• SDS sample buffer
• PMSF

Method including controls

1. Grow up overnight culture of bacteria to be induced.
2. Pipette 1ml of culture into a conical flask containing 100ml of LB and selective antibiotic.
3. Culture the bacteria at 25 (or 37) degrees C with shaking till the optical density at 600nm reaches about 1.
4. Add IPTG to a final concentration of 100uM.
5. Continue to culture the bacteria for 2-4 hours (depending on the protein).
6. Centrifuge at about 8000g for 10 minutes.
7. Discard the supernatant. Place on ice.
8. Resuspend using a pipette in 5ml of cold 1 X PBS (or TBS)
9. Add 5ul of PMSF
10. Sonicate 2 or 3 times in 15 second bursts with about 2 minutes rest in between bursts (keep the tube on ice at all times- especially between bursts).
11. Add Triton X-100 to a final volume of 1%. Mix gently for 30 minutes e.g. on a rocker.
12. Centrifuge for 10 minutes at 12,000g.
13. Save both the supernatant and the pellet.
    -Transfer the supernatant to a new tube. This is the soluble fraction.
    -Resuspend the pellet in 1ml of 1XPBS. This is the insoluble fraction.
    Both fractions can be frozen at this point if down-stream applications involve denaturing the protein (e.g. using SDS).
14. To 1ml of the above fractions (i.e. 1ml from the supernatant and all of the pellet re-suspention) add SDS sample (loading) buffer to make 1X buffer.
15. Heat at 100 degrees for 10 minutes. This is your protein sample ready to load on SDS-PAGE. They can be safely stored in the freezer.

Equipement Required

• Pipette and tips
• Conical flask
• Spectrophotometer
• Plastic Cuvettes
• Shaker
• Centrifuge
• ice
• Sonicator
• Heat block

References