Isolation and Characterization of Lipopolysaccharides - Bacterial Pathogenesis: Methods and Protocols - page 8

5. Solubilize the LPS or LOS in sample buffer to the desired concentration (0.1-1
mg/mL should be sufficient). Dilute 1:1 in dye buffer, boil for 5 min in a water
bath, and allow to cool. Load 5-10 μL per well.
6. To electrophorese, remove the comb and rinse the sample wells with reservoir
buffer. Fill the wells with reservoir buffer and add samples allowing them to
sink to the bottom. Place gels into the chamber and electrophorese at constant
current under the following conditions; for one 12 × 14 cm slab gel, use 10-12
mA through the spacer gel and then raise to 15 mA through the resolving gel.
Total run time will be approximately 5 h. For two 12 × 14 cm slab gels, use 20
mA through spacer gel and then raise to 30 mA through resolving gel. The total
run time will be 5-6 h.
7. Stain the gels using the silver stain method described by Tsai and Frasch (21) or
prepare the gels for western blotting (22) .
3.5. Silver Staining Technique for LPS and LOS Gels (23)
LPS and LOS bands can be rapidly identified in acrylamide gels using the
silver stain method of Tsai et al. (21) . This method is very sensitive and requires
careful attention to cleanliness of the glassware used. Only a glass tray should
be used for the procedure, and it is best to have dedicated trays for each step in
the procedure. Glassware should be pre-cleaned before each study with nitric
acid and washed thoroughly with distilled water. Gloves should be worn while
performing the procedure. Mild rotary agitation (70 rpm) on a platform rocker
is required for each step. The rinses and washes are crucial and should not be
reduced in time or number.
1. As soon as the electrophoresis is complete, place the gel in a fixing solution
consisting of 40% ethanol and 5% acetic acid in distilled water overnight. Add
0.9% periodic acid (1.8 g/200 mL) to fixing solution.
2. Rinse three times with distilled water. Transfer to a separate dish and wash three
additional times with distilled water (500-1000mL), agitating for 10min each time.
3. In a separate dish, pour in freshly prepared staining reagent (this should be made
during the last distilled water wash). This is made by mixing in the following
order in a hood, 28 mL of 0.1 N NaOH, 2.1 mL of concentrated NH 4 OH,5mL
of 20% silver nitrate (add silver nitrate drop-wise), and 115 mL distilled water.
4. Agitate for 10 min. Transfer the gel to a separate dish and rinse gel three times
with distilled water.
5. Transfer gel to a separate dish and add fresh formaldehyde developer (50 mg
anhydrous citric acid, 0.5 mL of 37% formaldehyde). For best results, the gel
should be developed in the dark using a Kodak GBX-2 safelight.
6. The bands will develop over the next 10-15 min. Background staining will
intensify in proportion to the amount of time the gel is left in the developer. Stop
the reaction by rinsing the gel in water, transferring to a separate dish and adding
rapid fix (10 mL in 100 mL of distilled water).