12 min at a temperature above 68
C with slow agitation
(hence insuring proper phenol/water miscibility).
3. Separate the different phases of the extraction by centrifuging
at 3345 Â g for 15 min at room temperature. Such centrifugation should result in obtaining three distinct phases of various
densities. The lighter, whiter solution is the aqueous phase,
whereas the heavier, yellower solution is the phenol phase in
which the leptospiral LPS are expected. Both phases are separated by a white interface containing denaturated proteins.
Membrane fragments and a residual pellet of non-lysed bacteria
might remain visible at the bottom of the tube (see Note 10).
4. Recover the aqueous and phenol phases without contamination by the white interface and the bacterial debris (see Notes
11 and 12).
Fig. 2 Schematic protocol of the classical hot water/phenol extraction purification described by Westphal et al.
[6] adapted for leptospiral LPS extraction
58
Delphine Bonhomme and Catherine Werts
C with slow agitation
(hence insuring proper phenol/water miscibility).
3. Separate the different phases of the extraction by centrifuging
at 3345 Â g for 15 min at room temperature. Such centrifugation should result in obtaining three distinct phases of various
densities. The lighter, whiter solution is the aqueous phase,
whereas the heavier, yellower solution is the phenol phase in
which the leptospiral LPS are expected. Both phases are separated by a white interface containing denaturated proteins.
Membrane fragments and a residual pellet of non-lysed bacteria
might remain visible at the bottom of the tube (see Note 10).
4. Recover the aqueous and phenol phases without contamination by the white interface and the bacterial debris (see Notes
11 and 12).
Fig. 2 Schematic protocol of the classical hot water/phenol extraction purification described by Westphal et al.
[6] adapted for leptospiral LPS extraction
58
Delphine Bonhomme and Catherine Werts