Biology Reference
In-Depth Information
Place this mixture at 37 °C for4htoallow digestion of any residual contaminating
nucleic acids.
6. The final step is a phenol extraction of the LPS nuclease-treated mixture to assure
removal of all residual protein contamination. Place the LPS mixture in a 65 °C
water bath for 30 min, add an equal volume of 90% phenol preheated to 65 °C,
and allow to set at 65 °C for 15 min. Place the mixture in an ice bath and cool
quickly to 4 °C.
7. Centrifuge the cooled mixture at 6000 × g for 15 min. Remove the aqueous top
layer and re-extract the phenol layer with an equal volume of distilled water. Heat
sample again to 65 °C for 15 min and then place in ice water. After centrifugation
at 6000 × g for 15 min, add top aqueous layer to the first aqueous extraction, and
dialyze against multiple changes of distilled water over 2 days. Discard the bottom
phenol layer. After dialysis, the LPS can be lyophilized and stored indefinitely at
room temperature.
3.3. Small-Scale Preparations of LPS or LOS
Occasionally, analytical studies such as western blot analysis of LPS or LOS
will require preparation of same quantities of these glycolipids from multiple
bacterial samples. Two methods have been described which can be used for
such isolations (18,19) . One relies on a modified phenol-water extraction (20) ,
and the second utilizes SDS solubility of bacteria followed by enzymatic degra-
dation of bacterial proteins (19) . Neither preparation gives a highly purified
preparation, but both enrich for LPS or LOS and can serve as adequate substi-
tutes in acrylamide gel and western blot studies. These two methods are
presented below.
3.3.1. Rapid Isolation Micro Method for LPS (20)
1. Centrifuge a bacterial suspension (10 8 colony-forming units/mL in 2 mL PBS)
in a 15-mL tube at 10,000 × g for 5 min. Wash the pellet once in PBS (pH 7.2)
containing 0.15 m M CaCl 2 and 0.5 m M MgCl 2 . Resuspend washed cells in 300
μL of water and transfer to a 1-dram vial containing a stir bar.
2. Add an equal volume of hot (65-70 °C) 90% phenol. Stir the mixture vigorously
at 65-70 ° for 15 min. Chill suspension on ice, transfer to a 1.5-mL polypropylene
tube, and centrifuge at 8500 × g for 15 min.
3. Transfer supernate to a 15-mL conical centrifuge tube. Re-extract phenol phase
with 300 μL of distilled water. Pool the aqueous phases.
4. Add sodium acetate to 0.5 M final concentration. Add 10 volume of 95% ethanol
and place sample at -20 °C overnight in order to precipitate the LPS. Centrifuge
precipitate at 2000 × g at 4 °C for 10 min. Discard supernatant.
 
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