Characterization of Lipopolysaccharides: Gel Filtration, Ion-Exchange, Capillary Electrophoresis and Micro-purification of LPS
Abstract
Immune cells identify lipopolysaccharides, which are abundant on the surface of most Gram-negative bacteria, as a chemical linked with pathogens. They are known as endotoxins because of their ability to induce serious infections such as sepsis. Lipopolysaccharide is composed of O-antigen repeats, core oligosaccharide, and lipid A. One of endotoxin's primary bioactivities is lipid A. Purifying and analysing lipopolysaccharides is challenging due to their unique structure and amphipathic characteristic. Lipopolysaccharide extraction, purification, and analysis methods are reviewed in this chapter. Numerous investigations aimed at isolating and purifying LPS were undertaken after its very important causal involvement in diseases caused by gramme negative bacteria was recognised. So it should come as no surprise that numerous protocols and methods have been developed for the separation and purification of LPS from bacteria. Some of them include trichloroacetic acid extraction at 4ºC, aqueous butanol, triton/Mg+2, cold ethanol, and water extraction at 100ºC. Specific procedures for the purification of raw LPS using phenol, chloroform, petroleum-ether, and methanol have also been detailed. There has been recent success in purifying LPS from E. coli using a combination of sized exclusion chromatography and the Westphal method, which is based on the hot phenol extraction procedure. Due to its high yield, this technique is most often used for LPS extraction. One major drawback of certain suggested LPS purification techniques is the possibility of protein and nucleic acid contamination, which would make it impossible to trust the final product in sensitive assays like molecular and immunological tests. While ultracentrifugation can be used to remove contaminants’ proteins, it typically results in decreased yields and a significant proportion of nucleic acids contaminating the sedimented LPS.
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