3.4.2 Qualitative Analysis
by LPS Adapted Silver
Staining
In order to visualize the LPS obtained after hot water/phenol
extraction, it is recommended to perform a silver staining coloration of the LPS after SDS-PAGE analysis (Fig. 3). Tsai and Frasch
have described a very sensitive silver staining method for LPS
visualization [8]. This protocol includes a specific oxidation step
with periodic acid that allows for a better coloration of the LPS (see
Note 18 for another method).
1. Perform the migration of the LPS samples boiled in Laemmli
buffer (1:1 dilution) on SDS-PAGE using a gradient mini-gel
4–15%. Recommended amount of LPS per well is between
100 ng and 10 μg. Migrate at 80 V for 1–1.5 h (see Note 19).
2. Fix the LPS sample by incubating the gel in the fixation solution overnight at room temperature without agitation.
3. Perform the oxidation step by transferring the gel in oxidation
solution for 5 min at room temperature and under slow agitation (40 rpm) (see Note 20).
4. Wash thoroughly the gel with ultrapure water (three times
15 min in 50 mL of water).
5. Prepare the staining solution: first, mix 7 mL of 1 N NaOH to
500 μL of ammonium hydroxide; then, add 1.25 mL of 20%
silver nitrate and finally add 28.75 mL of ultrapure water (see
Note 21). Incubate the gel in the coloration solution for
Fig. 3 Silver staining of SDS-PAGE (4–15%) of (a) various bacterial lipopolysaccharides, Leptospira interrogans serovar Icterohaemorrhagiae strain Verdun (lane 1), Escherichia coli O111:B4 (lane 2), and Salmonella
enterica serotype Minnesota (lane 3), and (b) comparison between the classical hot water/phenol extraction
(lane 1) and the fast proteinase K preparation (lane 2) of Leptospira interrogans serovar Icterohaemorrhagiae
strain Verdun LPS
60
Delphine Bonhomme and Catherine Werts
by LPS Adapted Silver
Staining
In order to visualize the LPS obtained after hot water/phenol
extraction, it is recommended to perform a silver staining coloration of the LPS after SDS-PAGE analysis (Fig. 3). Tsai and Frasch
have described a very sensitive silver staining method for LPS
visualization [8]. This protocol includes a specific oxidation step
with periodic acid that allows for a better coloration of the LPS (see
Note 18 for another method).
1. Perform the migration of the LPS samples boiled in Laemmli
buffer (1:1 dilution) on SDS-PAGE using a gradient mini-gel
4–15%. Recommended amount of LPS per well is between
100 ng and 10 μg. Migrate at 80 V for 1–1.5 h (see Note 19).
2. Fix the LPS sample by incubating the gel in the fixation solution overnight at room temperature without agitation.
3. Perform the oxidation step by transferring the gel in oxidation
solution for 5 min at room temperature and under slow agitation (40 rpm) (see Note 20).
4. Wash thoroughly the gel with ultrapure water (three times
15 min in 50 mL of water).
5. Prepare the staining solution: first, mix 7 mL of 1 N NaOH to
500 μL of ammonium hydroxide; then, add 1.25 mL of 20%
silver nitrate and finally add 28.75 mL of ultrapure water (see
Note 21). Incubate the gel in the coloration solution for
Fig. 3 Silver staining of SDS-PAGE (4–15%) of (a) various bacterial lipopolysaccharides, Leptospira interrogans serovar Icterohaemorrhagiae strain Verdun (lane 1), Escherichia coli O111:B4 (lane 2), and Salmonella
enterica serotype Minnesota (lane 3), and (b) comparison between the classical hot water/phenol extraction
(lane 1) and the fast proteinase K preparation (lane 2) of Leptospira interrogans serovar Icterohaemorrhagiae
strain Verdun LPS
60
Delphine Bonhomme and Catherine Werts