indirect dosage does not allow a real quantification but can be
valuable to compare several LPS purifications.
16. The glass tubes used for lyophilization should be detoxified at
256
C for at least 4 h in order to get rid of any potential
endotoxin traces that could contaminate the leptospiral LPS
preparation.
17. On average, a 7-day-old stationary culture of 2.5 L that had
been inoculated at around 10
6 leptospires/mL will give a
1–2 Â 10
12 bacterial pellet. Such bacterial pellet should result
in the extraction of 1–10 mg of LPS, depending on the strain
and the quality of the extraction.
18. The bacterial lipopolysaccharides absorb light at the wavelength of 190 nm. However, the heterogeneity of the molecule
makes it impossible to quantify the amount of LPS present in
the preparation by measuring the absorbance. Despite these
limitations, UV spectrum can be used to assess the quality of
the extraction in terms of nucleic acid (260 nm) and protein
(280 nm) contaminations.
19. Prefer the use of 10-well gels rather than 15-well gels, on
which the migration of the LPS is often altered by the narrowness of the well.
20. Throughout the silver staining analysis, it is important to avoid
touching the gel with metal component or with hands, because
that will leave traces visible during the revelation of the coloration. Only touch the edges of the gel with wet gloves.
21. It is very important to respect the order of addition of the
components of the solution. Also, note that a brown precipitate will form upon addition of the silver nitrate but should
disappear under gentle homogenization.
22. Be careful not to over incubate the gel in the revelation solution; otherwise the background coloration will lower the quality of the visualization.
23. In general, LPS of various bacteria such as Escherichia coli are
characterized by numerous bands of various sizes. However, in
the case of Leptospira interrogans strain Verdun, the LPS only
possess two major bands expected around 25 and 10 kDa, as
well as several minor bands (Fig. 3a). Other strains of
L. interrogans such as Manilae L495 and Copenhageni Fiocruz
L1–130 have similar LPS with minor variations in the size and
intensity of the bands. In addition, the differences in purity
between the LPS extracted with the fast proteinase K method
and the LPS purified with the classical method are visible on
silver staining analysis (Fig. 3b).
64
Delphine Bonhomme and Catherine Werts
valuable to compare several LPS purifications.
16. The glass tubes used for lyophilization should be detoxified at
256
C for at least 4 h in order to get rid of any potential
endotoxin traces that could contaminate the leptospiral LPS
preparation.
17. On average, a 7-day-old stationary culture of 2.5 L that had
been inoculated at around 10
6 leptospires/mL will give a
1–2 Â 10
12 bacterial pellet. Such bacterial pellet should result
in the extraction of 1–10 mg of LPS, depending on the strain
and the quality of the extraction.
18. The bacterial lipopolysaccharides absorb light at the wavelength of 190 nm. However, the heterogeneity of the molecule
makes it impossible to quantify the amount of LPS present in
the preparation by measuring the absorbance. Despite these
limitations, UV spectrum can be used to assess the quality of
the extraction in terms of nucleic acid (260 nm) and protein
(280 nm) contaminations.
19. Prefer the use of 10-well gels rather than 15-well gels, on
which the migration of the LPS is often altered by the narrowness of the well.
20. Throughout the silver staining analysis, it is important to avoid
touching the gel with metal component or with hands, because
that will leave traces visible during the revelation of the coloration. Only touch the edges of the gel with wet gloves.
21. It is very important to respect the order of addition of the
components of the solution. Also, note that a brown precipitate will form upon addition of the silver nitrate but should
disappear under gentle homogenization.
22. Be careful not to over incubate the gel in the revelation solution; otherwise the background coloration will lower the quality of the visualization.
23. In general, LPS of various bacteria such as Escherichia coli are
characterized by numerous bands of various sizes. However, in
the case of Leptospira interrogans strain Verdun, the LPS only
possess two major bands expected around 25 and 10 kDa, as
well as several minor bands (Fig. 3a). Other strains of
L. interrogans such as Manilae L495 and Copenhageni Fiocruz
L1–130 have similar LPS with minor variations in the size and
intensity of the bands. In addition, the differences in purity
between the LPS extracted with the fast proteinase K method
and the LPS purified with the classical method are visible on
silver staining analysis (Fig. 3b).
64
Delphine Bonhomme and Catherine Werts