LPS Isolation (Darveau-Hancock Method)
Categorized: Outer Membranes
- 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
- 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
- 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.)
- Resuspend 500 mg dried bacterial cells in 15 ml of 10 mM Tris-HCl, pH 8.0, 2 mM MgCl2.
- Add DNase (100 ug/ml) and RNase (25 ug/ml).
- French press the cell suspension twice at 15,000 psi.
- Sonicate for two 30s bursts at a probe intensity of 75.
- Add DNase and RNase to bring their final concentrations to 200 ug/ml and 50 ug/ml respectively.
- Incubate the suspension at 37oC for 2 hours.
- 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.
- Vortex and centrifuge at 50,000 g for 30 minutes at 20oC to remove peptidoglycan.
- Save supernatant. Add pronase to give a final concentration of 200 ug/ml.
- Incubate at 37oC with constant shaking overnight.
- Add two volumes of 0.375 M MgCl2/95% EtOH. Mix and cool to 0oC in -20oC refrigerator.
- After the sample had cooled to 0oC, centrifuge at 12,000 g for 15 minutes at 0 – 4oC.
- Resuspend pellet in 25 ml of 0.1 M EDTA(tetra sodium salt), 2% SDS, 10 mM Tris-HCl, pH 8.0.
- Sonicate at a probe intensity of 75 for two 30 s bursts.
- Incubate the solution at 85oC for 30 minutes. Cool to room temperature. Bring pH to 9.5 by addition of 4M NaOH.
- Add pronase to give a final concentration of 25 ug/ml. Incubate at 37oC overnight with constant shaking.
- Add two volumes of 0.375 M MgCl2/95% EtOH and cool solution to 0oC as before.
- Centrifuge at 12,000 g for 15 minutes at 0 – 4oC.
- 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.
- 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.
- Add MgCl2 to give a final concentration of 25mM. Centrifuge at 200,000 g (45K) for two hours.
- Resuspend pellet in distilled water. Lyophilize if necessary.
- Do KDO assay.
CHCl3:MeOH (2:1) Treatment (optional)
- Add equal volume of CHCl3:MeOH (2:1) to sample. Vortex.
- Centrifuge at 9K for 10 minutes.
- Discard the bottom layer (retain white precipitate).
- Repeat the extraction (step 1 – 3).
- Drive reminiscent CHCl3 and MeOH by vacuum suction or lyophilize sample.
Lipopolysaccharide Of Sodium Salt
- 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.
- Lyophilize sample.
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