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- PROTOCOL] Applications of different solvents and conditions for differential extraction of lipopolysaccharide in Gram-negative bacteria
- Mai Phuong Nguyen 1, Le Viet Ha Tran 1, Hyun Namgoong 2, Yong-Hak Kim 1
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Journal of Microbiology 2019;57(8):644-654.
DOI: https://doi.org/10.1007/s12275-019-9116-5
Published online: May 23, 2019
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Corresponding author:
Yong-Hak Kim , Tel: +82-53-650-4338,
Abstract
Lipopolysaccharide (LPS) is one of the major components in
the outer membrane of Gram-negative bacteria. However,
its heterogeneity and variability in different bacteria and differentiation
conditions make it difficult to extract all of the
structural variants. We designed a solution to improve quality
and biological activity of LPS extracted from various bacteria
with different types of LPS, as compared to conventional
methods
. We introduced a quality index as a simple measure
of LPS purity in terms of a degree of polysaccharide content
detected by absorbance at 204 nm. Further experiments using
gel electrophoresis, endotoxin test, and macrophage activation
test were performed to evaluate the performance and reliability
of a proposed ‘T-sol’ method and the biological effectiveness
and character of the LPS products. We presented
that the T-sol method had differential effects on extraction of
a RAW 264.7 cell-activating LPS, which was effective in the
macrophage activation with similar effects in stimulating
the production of TNF-alpha. In conclusion, the T-sol method
provides a simple way to improve quality and biological activity
of LPS with high yield.
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