Biology Reference
In-Depth Information
3. Wash the sample again with 500 μL of non-acidified 95% ethanol and centrifuge
at 2000 × g for 10 min.
4. Repeat centrifugation and washing steps. Finally, lyophilize the sample to yield
fluffy white solid lipid A (26) .
4. Notes
1. Occasionally, the suspension will become gelatinous at this stage. If this occurs,
stir for 3 min in the blender before phenol extraction.
2. The dialyzate is still a relatively crude LPS preparation that needs further purifi-
cation by centrifugation. This will remove residual nucleic acids and any capsular
materials that remain in the sample. The preparation is suspended in distilled water
at concentrations of 25-35 mg/mL. At times, this crude LPS can be difficult to
suspend. It should be vortexed vigorously to obtain a smooth suspension. Gentle
sonication may be required to obtain an even distribution of the LPS suspension.
3. Purity of the LPS and LOS preparations: After isolation of the LOS or LPS, the
degree of purity should be ascertained. Nucleic acid and protein contamination
are the principle concerns. Spectral analysis from 245 nm thru 290 nm using
purified LPS or LOS at 1 mg/mL can be performed to determine nucleic acid
and protein contamination. Agarose gel electrophoresis with ethidium bromide
staining can also be useful as a highly sensitive means of determining the degree
of nucleic acid contamination. Acrylamide gel electrophoresis of the LOS/LPS
preparation will define the physical characteristics of the preparation (21) . This
method utilized in a number of laboratories studying LPS and LOS is described
in Subheading 3.4 . The pore size of the gel can be adjusted according to the
preparation under study and can range from 10 to 16% acrylamide.
References
1. Shear, M. J. (1941) Effect of concentrate from B. prodigiosus filtrate on subcuta-
neous primary induced mouse tumors. Cancer Res . 1 , 732-741.
2. Luderitz, O., Staub, A. M., and Westphal, O. (1966) Immunochemistry of O
and R antigens of Salmonella and related Enterobacteriaceae . Bacteriol. Rev . 30 ,
192-255.
3. Goldman, R. C. and Leive, L. (1980) Heterogeneity of antigenic-side-chain length
in lipopolysaccharide from Escherichia coli 0111 and Salmonella typhimurium
LT2. Eur. J. Biochem . 107 , 145-153.
4. Palva, E. T. and Makela, P. H. (1980) Lipopolysaccharide heterogeneity in
Salmonella typhimurium analyzed by sodium dodecyl sulfate/polyacrylamide gel
electrophoresis. Eur. J. Biochem . 107, 137-143.
5. Preston, A., Mandrell, R. E., Gibson, B. W., and Apicella, M. A. (1996) The
lipooligosaccharides of pathogenic gram-negative bacteria. Crit. Rev. Microbiol .
22 , 139-180.
 
Search WWH ::




Custom Search