Lab Resources

Enzyme Expression Studies: Gel Sample Prep for SDS-Gels (Version 1-10-2010)

Set up a series of 3 Sample Tubes at room temperature for each time point sample:

  • TCP = 'Total Cell Protein'
  • SOL = 'Soluble Protein'
  • INS = 'Insoluble Protein'
Each tube should contain:
  • 15 µl 10% glycerol
  • 30 µl 2x SDS Sample Buffer
  1. Keep culture aliquots on ice. Pellet sufficient volume for each time point to be equivalent to 6 A600 Units of cells. For example, if the culture OD was 3.5, then the volume of culture to yield 6 OD's of cells would be: 6 OD's/(3.5 OD) = 1.71 ml. Usually a 2.0 ml microfuge tube is sufficient even if it requires multiple additions/pelleting/aspiration and so on to reach 6 OD's. Ensure that ALL of the culture fluid has been removed. Aspiration is the best method. Culture samples should be spun at 6,000 rpm in a microcentrifuge for 5 minutes.
  2. Add 200 µl Lysis Buffer + Lysozyme to each pellet. (Lysis Buffer: 50 mM TRIS, pH 8; 2 mM EDTA, 300 µg lysozyme/ml). Vortex until the pellets have been uniformly resuspended.
  3. Incubate at 37 °C for 10 minutes and place on ice. The solutions should be very viscous due to the chromosomal DNA that is released upon lysis by lysozyme.
  4. Sonicate using the micro-tip (20% Duty Cycle, 0% Output) for 30 to 40 bursts taking care to ensure that the tip is always under the level of the solution in the tube.
  5. The samples now should be nearly as fluid as water because the sonication shears the chromosomal DNA. Add 22.4 µl 5 M NaCl to each tube, seal, and vortex.
  6. Remove 15 µl of the sonicate and transfer it to the appropriate 'TCP' sample tube alread containing the Sample Buffer + glycerol.
  7. Spin the remainder of the sonicate in a microcentrifuge at 13,000 rpm for 10 minutes at room temperature.
  8. Remove 15 µl of the supernatant to the appropriate 'SOL' sample tube already containing the Sample Buffer + glycerol.
  9. Aspirate off the remaining supernatant.
  10. Add 400 µl Lysis Buffer with 500 mM NaCl and resuspend the pellet using the same sonication procedure as in step #4. This is a 'wash step' to remove any residual soluble protein from the pellet and from the walls of the tube.
  11. Spin the suspension at 13,000 rpm for 10 minutes at room temperature.
  12. Aspirate off and discard the supernatant.
  13. Add 192 µl of Lysis Buffer and resuspend the pellet by sonication as in step #4. Usually only 10 to 12 bursts are necessary to resuspend the pellet.
  14. Remove 15 µl of the resuspended pellet solution to the appropriate 'INS' tube.
  15. Heat all sample tubes at 80-85 °C for 5 minutes prior to loading 10-15 µl per lane for SDS PAGE.

Run gels at 100 VDC until the bromophenol reaches the bottom of the running gel.

Protocol contributed by John Brandis, Ph.D.

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This page last updated Thu, February 11, 2010 11:42