Recovery of LPS for SDS-PAGE

  1. Inoculate 1-2 ml of L-broth or Terrific broth supplemented with the appropriate antibiotics with the bacteria to be tested. Grow cultures several hours at 37°C on a roller to mid-log phase.

  2. Pipette 0.5 ml of the culture to L-agar plates containing appropriate antibiotics. Spread aliquot over plate using a glass spreader. Incubate plates upright at 37°C overnight.

  3. Pipette 3 ml of 1x PBS buffer onto plate. Suspend bacterial lawn with a glass spreader. Tilt the plate, collect the suspension with a transfer pipette and place in glass culture tubes.

  4. Autoclave tubes for 30 minutes at 121°C.

  5. Agitate the cells to liberate LPS by vortexing or vigorous shaking for several minutes. Transfer the suspension to appropriately-sized centrifuge tubes.

  6. Pellet the cells by centrifugation at 2,500 rpm for 15 minutes at 4°C. Decant supernatant to 50-ml polypropylene conical tubes.

  7. Add four volumes of ice-cold 95% ethanol to the supernatant. Mix and precipitate overnight at -20°C.

  8. Collect the precipitate by centrifugation for 15 minutes at 2,500 rpm at 4°C. Dry the pellet with tubes inverted over paper towels.

  9. Dissolve the dried pellet in a minimal amount of sterile 1x PBS (i.e. 0.5 ml). Transfer suspension to 1.7-ml microcentrifuge tubes.

  10. Remove an aliquot of the samples sufficient for analysis by SDS-PAGE. Digest sample with DNAse and RNAse (both at 100 µg/ml) at 37°C overnight. The remainder of the sample can be held at -20°C.

  11. Digest the sample with pronase (100 µg/ml) for 2 hours at 56°C.

  12. Combine the sample with an equal volume of 2x sample buffer. Incubate at 65°C for 30 minutes.

  13. The samples are now ready for SDS-PAGE analysis. They may be held at -20°C.

2x Sample Buffer

4x Tris-Cl/SDS, pH 6.8     25.0 ml
glycerol                   20.0 ml
SDS (electrophoresis-grade) 4.0 g
DTT                         3.1 g
bromphenol blue             1.0 mg

Combine reagents in a final volume of 100 ml dH2O. Store in 1 ml aliquots at -70°C.

The resulting mixture is as follows (at 2x):

Tris              0.125 M
SDS               4.1 %
glycerol         20.0 %
DTT               3.1 %
bromphenol blue   0.001 %
Terrific Broth

bacto-tryptone        12.0 g
bacto-yeast extract   24.0 g
glycerol               4.0 ml
Combine reagents in 900 ml dH2O (final volume) and autoclave.
Before using, add 100 ml of 0.17 M KH2PO4 and 0.72 M K2HPO4 solution prepared as follows:

KH2PO4   2.31 g
K2HPO4  12.54 g
Dissolve reagents in 90 ml dH2O. Adjust volume to 100 ml. Sterilize by autoclaving.

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