Hancock Lab

LPS Isolation (Darveau-Hancock Method)

Categorized: Outer Membranes

Reference:

J. Bacteriol. 155: 831-838. (Darveau, Hancock method)

Reagents :

  • 10mM Tris-HCl, pH 8.0
  • 10 mM Tris-HCl, pH 8.0; 2 mM MgCl2
  • 0.5 M EDTA in 10 mM Tris-HCl, pH 8.0
  • 20% SDS in 10 mM Tris-HCl, pH 8.0
  • 0.375 M MgCl2 in 95% EtOH
  • 0.1 M EDTA, 2% SDS, 10 mM Tris-HCl, pH 8.0
  • Deoxyribonuclease I
  • Ribonuclease
  • Protease/Pronase

Methods:

  1. Grow 1 litre of bacterial cell in an appropriate media (Proteose Peptone No. 2 for P. aeruginosa) until an O.D. 600 nm of 0.6 – 0.8
  2. Harvest cells at 7,000 rpm for 15 minutes. Lyophilize. (Note : 1 litre of wet bacterial cells equivalent to 500 mg of dried bacterial cells.)
  3. Resuspend 500 mg dried bacterial cells in 15 ml of 10 mM Tris-HCl, pH 8.0, 2 mM MgCl2.
  4. Add DNase (100 ug/ml) and RNase (25 ug/ml).
  5. French press the cell suspension twice at 15,000 psi.
  6. Sonicate for two 30s bursts at a probe intensity of 75.
  7. Add DNase and RNase to bring their final concentrations to 200 ug/ml and 50 ug/ml respectively.
  8. Incubate the suspension at 37oC for 2 hours.
  9. Add 5 ml of 0.5 M EDTA(tetra sodium salt)/10 mM Tris, pH 8.0; 2.5 ml of 20% SDS/10 mM Tris, pH 8.0 and 2.5 ml of 10 mM Tris-HCl, pH 8.0 to give a final volume of 25 ml, final pH approx. 9.5.
  10. Vortex and centrifuge at 50,000 g for 30 minutes at 20oC to remove peptidoglycan.
  11. Save supernatant. Add pronase to give a final concentration of 200 ug/ml.
  12. Incubate at 37oC with constant shaking overnight.
  13. Add two volumes of 0.375 M MgCl2/95% EtOH. Mix and cool to 0oC in -20oC refrigerator.
  14. After the sample had cooled to 0oC, centrifuge at 12,000 g for 15 minutes at 0 – 4oC.
  15. Resuspend pellet in 25 ml of 0.1 M EDTA(tetra sodium salt), 2% SDS, 10 mM Tris-HCl, pH 8.0.
  16. Sonicate at a probe intensity of 75 for two 30 s bursts.
  17. Incubate the solution at 85oC for 30 minutes. Cool to room temperature. Bring pH to 9.5 by addition of 4M NaOH.
  18. Add pronase to give a final concentration of 25 ug/ml. Incubate at 37oC overnight with constant shaking.
  19. Add two volumes of 0.375 M MgCl2/95% EtOH and cool solution to 0oC as before.
  20. Centrifuge at 12,000 g for 15 minutes at 0 – 4oC.
  21. Resuspend pellet in 15 ml of 10 mM Tris-HCl, pH 8.0. Sonicate at a probe intensity of 75 for two 30 s bursts.
  22. Centrifuge at 1000 rpm for 5 minutes to remove insoluble Mg/EDTA complexes.
    Step 22a. For rough LPS, wash the pellet in small volume of water; recentrifuge and add the supernatant to the outer supernatant from Step. 22.
  23. Add MgCl2 to give a final concentration of 25mM. Centrifuge at 200,000 g (45K) for two hours.
  24. Resuspend pellet in distilled water. Lyophilize if necessary.
  25. Do KDO assay.

CHCl3:MeOH (2:1) Treatment (optional)

  1. Add equal volume of CHCl3:MeOH (2:1) to sample. Vortex.
  2. Centrifuge at 9K for 10 minutes.
  3. Discard the bottom layer (retain white precipitate).
  4. Repeat the extraction (step 1 – 3).
  5. Drive reminiscent CHCl3 and MeOH by vacuum suction or lyophilize sample.

Lipopolysaccharide Of Sodium Salt

  1. Dialyse the above CHCl3:MeOH treated LPS with:
    0.5 mM Hepes, pH 7.4, 5 mM Na2EDTA, pH 8.0. (five times)
    5mM Hepes, pH 7.4, 50 mM NaCl. (two times)
    Distilled water (two times)
    Change dialysate every 8 hours.
  2. Lyophilize sample.

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