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

• polyacrylamide gel
• 1X SDS buffer (6.05g Tris, 28.83g glycine, 2g SDS per liter)
• Benchmark Protein Ladder
• Denatured protein samples
• Commassie blue (50% methanol, 10% acetic acid, 0.2% commassie)
• Destain solution (10% methanol, 10% acetic acid)

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 degrees C till the optical density at 600nm reaches about 1.
4. Add IPTG to a final concentration of 1mM.

1. Centrifuge at about 8000g=8000rpm for 10 minutes. 2. Discard the supernatant. Place on ice. 3. Resuspend using a pipette in 5ml of cold 1 X PBS (or TBS) 4. 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).

5. Add Triton X-100 (this can be changed to a different detergent depending on what you want to solubilize- but I suggest you try this first) to a final volume of 1%. Mix gently for 30 minutes on a wheel or rocker or whatever is available.

6. Centrifuge for 10 minutes at 10,000rpm=12,000g. 7. 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 as is in this case).

8. 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 1Xbuffer. This is a blue buffer that you should ask for in the lab. The buffer is usually 6X in the original form so add 200 microliters of it to your fractions.

9. 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.

10. Optional. You can do a Bradford assay to quantitate the amount to protein you have in each sample to equalize the amount of protein in each lane. Personally for what you are doing now I don't think this is necessary. I advise to just add 10 microliters of each sample to the gel.

Equipement Required

• Pipette and tips
• SDS PAGE apparatus
• Power source

References