Biology Reference
In-Depth Information
5. Suspend pellet in 100 μL of distilled water and transfer to a 1.5 mL polypropylene
tube. Repeat ethanol precipitation. Dry the precipitate and resuspend with 50 μL
of distilled water. Store at -20 °C indefinitely.
3.3.2. LPS Microextraction Using Proteinase K Digestion (19)
1. Harvest organisms grown on solid medium with a sterile swab and suspend in
10 mL of cold phosphate-buffered saline (PBS) (pH 7.2) to a turbidity of 0.4
absorbance at 650 nm. Centrifuge a portion (1.5 mL) of this suspension for 1.5
min in a microfuge at 14,000 × g .
2. Solubilize the pellet in 50 μL of lysing buffer containing 2% SDS, 4%
2-mercaptoethanol, 10% glycerol, 1 M Tris-Cl (pH 6.8), and bromophenol blue.
Heat sample at 100 °C for 10 min.
3. To digest bacterial proteins, add 25 μg of proteinase K in 10 μL of lysing buffer
to each boiled lysate and incubate at 60 °C for 60 min.
4. Use the preparation in acrylamide gel electrophoresis or for western blots in
volumes ranging from 0.5 to 2 μL ( see Note 3 ).
3.4. LOS and LPS Acrylamide Gel Electrophoresis
Acrylamide gel electrophoresis of LOS and LPS can be an exasperating
procedure because of day-to-day variation in resolution. To ensure consistency
of these gels, acid wash all glassware and rinse well before drying; all reagents
must be of the highest quality, and the quality of water should be in the 18 ohm
range. Finally, stock solutions should not be used past the time recommended.
1. To prepare two 14% resolving gels, combine 18.45 mL of 30% acrylamide,
7.9 mL of resolving buffer, and 12.57 mL of distilled water. Place solution in a
vacuum flask and degas for 15 min.
2. Add 0.3 mL ammonium persulfate solution and N,N,N´,N´-tetramethylethylene-
diamine (TEMED) (10 μL/50 mL gel solution). The resolving gel should be poured
between the glass plates as soon as the TEMED is added and the solution gently
mixed. Overlay the gel with 2 mm distilled water and allow to polymerize for at
least 2 h. This will make a total of 39.56 mL resolving gel, which is sufficient
for two 12 × 14 cm slab gels with a 0.75-mm spacer. Double the volumes if
you are using 1.5-mm spacers. To make 16% resolving gels, mix 10.55 mL of
30% acrylamide, 3.95 mL resolving buffer, and 4.95 mL of distilled water. The
quantities of the other reagents are unchanged.
3. To prepare the spacer gel, combine 2 mL of 30% acrylamide, 2 mL of spacer
buffer, and 15.6 mL of distilled water. Degas the solution for 15 min and add 0.2
mL of ammonium persulfate, 0.2 mL of 10% SDS, and 10 μL of TEMED. Mix
gently. The total volume of the spacer gel is 20.01 mL.
4. Remove the layer of distilled water over the resolving gel and pour the spacer
gel. Insert at this time the comb chosen for the run. Allow the spacer gel to
polymerize for at least 1 h but preferably overnight.
 
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