Isolation and Characterization of Lipopolysaccharides - Bacterial Pathogenesis: Methods and Protocols - page 4

2. Materials
2.1. LOS and LPS Acrylamide Gel Electrophoresis
1. 30% Acrylamide Stock: Dissolve 29.2 g acrylamide and 0.8 g bis-acrylamide in
40 mL of distilled water and q.s. to 100 mL. Filter into dark bottle and store at
4 °C. This stock should not be used after 2 weeks.
2. Resolving Buffer, 1.88 M Tris-HCl (pH 8.8): Dissolve 22.78 g Trizma base in
70 mL of distilled water, adjust pH to 8.8 with concentrated HCl and q.s. to 100
mL. Store at 4 °C. This stock should not be used after 2 weeks.
3. Spacer Buffer, 1.25 M Tris-HCl (pH 6.8): Dissolve 15.12 g Trizma base in 70
mL of distilled water, adjust pH to 6.8 with concentrated HCl and q.s. to 100 mL.
Store at 4 °C. This stock should not be used after 2 weeks.
4. Sample Buffer: Add 0.727 g Trizma base, 0.034 g EDTA, and 2.0 g SDS to 70
mL distilled water, adjust pH to 6.8 with concentrated HCl, q.s. to 100 mL with
distilled water, filter sterilize, and store at room temperature.
5. Dye Buffer: Combine 2.5 mL of sample buffer, 2.0 mL of glycerol, 400 μL of
-mercaptoethanol, and 200 μL of a saturated solution of bromophenol blue. This
buffer should be made fresh for each run. A stock solution can be made without
adding the -mercaptoethanol. This should be added just prior to use.
6. Ammonium Persulfate: Dissolve 0.05 g ammonium persulfate in 1 mL of distilled
water. This solution should be made fresh for each experiment.
7. Reservoir Buffer, Tris-glycine buffer (pH 8.3): Dissolve 115.2 g glycine, 24 g
Trizma base, and 8.0 g SDS in8Lofdistilled water. No pH adjustment should
be necessary if correct reagents and quantities are added.
3. Methods
3.1. Phenol-Water Technique (16)
1. Place5goffreeze- or acetone-dried bacteria in a mortar and pestle and ground
until a very fine powder is formed. Suspend powder in 25 mL of 50 m M sodium
phosphate (pH 7.0) containing 5 m M EDTA. Allow suspension to completely
hydrate before proceeding.
2. Stir suspension in a shearing mixer such as a blender at top speed for 1 min. Add
hen egg lysozyme (100 mg) to the suspension and stir overnight at 4 °C.
3. Place suspension at 37 °C for 20 min and then stir at top speed in a blender for 3
min. Increase volume of the suspension to 100 mL with 50 m M sodium phosphate
(pH 7.0) containing 20 m M MgCl 2 . Add ribonuclease A and deoxyribonuclease I
to a final concentration of 1 μg/mL. Incubate the suspension for 60 min at 37 °C
and then for 60 min at 60 °C ( see Note 1 ).
4. Place the bacterial uspension in a 70 °C water bath until the temperature equili-
brates. Add an equal volume of 90% (w/v) phenol that has been preheated to
70 °C and mixed thoroughly. The resulting mixture is rapidly cooled by stirring
for 15 min in an ice water bath.