Melbourne/Preparing Protein Samples for SDS PAGEl


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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: $


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