3.3.2 Dialysis and
Ultracentrifugation
1. In order to evacuate the phenol contaminant, perform dialysis
of the two phases recovered from the extraction against large
volumes of hot tap water (minimum 68
C), using dialysis
membranes with a molecular weight cutoff of
12,000–14,000 Da. Slow agitation is recommended for a
more efficient dialysis (see Note 13). A minimum of 2–3 L
should be use to dialyze the water phase and minimum of
6–7 L for the phenol phase.
2. Perform an ultracentrifugation of 3 h, 100,000 Â g, 10
C to
remove nucleic acids and proteins from the LPS preparation.
3. Resuspend the white “flour-like” pellet obtained in large volume (25 mL minimum) of ultrapure endotoxin-free water. To
ensure proper removal of the contaminants, it is important to
vortex the LPS pellet thoroughly before processing to step 4.
4. Repeat steps 2 and 3 three times.
5. Resuspend the pellet obtained after the last step of ultracentrifugation in 1 or 2 mL of endotoxin-free water. This LPS
preparation can be used for quantitative and qualitative analysis
and can be lyophilized for weighting.
3.4 “Crude” LPS
Analysis
3.4.1 Quantification by
Lyophilization
Up to now, the best way to quantify the amount of LPS extracted
during the purification remains weighting after lyophilization. A
12 h lyophilization is in general sufficient to remove all the water
from LPS purifications. However, this method allows the quantification of the LPS mass but not molarity, since the LPS is composed
of a mixture of different molecules with various O antigen structures and sizes. Hence, this quantification (which is currently the
only available one) is strongly biased, and such limit must be taken
into consideration during experiments (see Notes 14 and 15 for
other methods).
1. Weight and tare an empty 2 mL endotoxin-free glass tube (see
Note 16).
2. Put the resuspended LPS sample in the tube and freeze it for at
least 3 h at À80
C.
3. Remove the cap and replace it with parafilm in which small
holes must be drilled. Conserve the cap in sterile environment.
4. Lyophilize the LPS for at least 12 h.
5. Recover the sample, close back the tube in a sterile environment, and weight the amount of LPS obtained (see Note 17).
6. Resuspend the LPS at concentration between 1 and 10 mg/
mL for long-term storage at À20 or À80
C.
Purification of LPS from Leptospira
59
Ultracentrifugation
1. In order to evacuate the phenol contaminant, perform dialysis
of the two phases recovered from the extraction against large
volumes of hot tap water (minimum 68
C), using dialysis
membranes with a molecular weight cutoff of
12,000–14,000 Da. Slow agitation is recommended for a
more efficient dialysis (see Note 13). A minimum of 2–3 L
should be use to dialyze the water phase and minimum of
6–7 L for the phenol phase.
2. Perform an ultracentrifugation of 3 h, 100,000 Â g, 10
C to
remove nucleic acids and proteins from the LPS preparation.
3. Resuspend the white “flour-like” pellet obtained in large volume (25 mL minimum) of ultrapure endotoxin-free water. To
ensure proper removal of the contaminants, it is important to
vortex the LPS pellet thoroughly before processing to step 4.
4. Repeat steps 2 and 3 three times.
5. Resuspend the pellet obtained after the last step of ultracentrifugation in 1 or 2 mL of endotoxin-free water. This LPS
preparation can be used for quantitative and qualitative analysis
and can be lyophilized for weighting.
3.4 “Crude” LPS
Analysis
3.4.1 Quantification by
Lyophilization
Up to now, the best way to quantify the amount of LPS extracted
during the purification remains weighting after lyophilization. A
12 h lyophilization is in general sufficient to remove all the water
from LPS purifications. However, this method allows the quantification of the LPS mass but not molarity, since the LPS is composed
of a mixture of different molecules with various O antigen structures and sizes. Hence, this quantification (which is currently the
only available one) is strongly biased, and such limit must be taken
into consideration during experiments (see Notes 14 and 15 for
other methods).
1. Weight and tare an empty 2 mL endotoxin-free glass tube (see
Note 16).
2. Put the resuspended LPS sample in the tube and freeze it for at
least 3 h at À80
C.
3. Remove the cap and replace it with parafilm in which small
holes must be drilled. Conserve the cap in sterile environment.
4. Lyophilize the LPS for at least 12 h.
5. Recover the sample, close back the tube in a sterile environment, and weight the amount of LPS obtained (see Note 17).
6. Resuspend the LPS at concentration between 1 and 10 mg/
mL for long-term storage at À20 or À80
C.
Purification of LPS from Leptospira
59