# A Simple and Rapid Microscale Method for Isolating Bacterial Lipopolysaccharides

## TL;DR

This paper introduces a quick and simple method to isolate bacterial lipopolysaccharides from small samples while preserving their structure and function.

## Contribution

A novel microscale method for LPS extraction that preserves structural and functional integrity from limited bacterial material.

## Key findings

- Extracted LPSs contained less than 2–3% protein and 1% nucleic acid.
- Structural integrity of O-antigen and lipid A was confirmed using SDS–PAGE and MALDI–MS.
- Extracted LPSs retained functional activity by inducing cytokine and chemokine secretion in macrophages.

## Abstract

Bacterial endotoxins (lipopolysaccharides (LPSs)) are important mediators of inflammatory processes induced by Gram-negative microorganisms. LPSs are the key inducers of septic shock due to a Gram-negative bacterial infection; thus, the structure and functions of LPSs are of specific interest. Often, highly purified bacterial endotoxins must be isolated from small amounts of biological material. Each of the currently available methods for LPS extraction has certain limitations. Herein, we describe a rapid and simple microscale method for extracting LPSs. The method consists of the following steps: ultrasonic destruction of the bacterial material, LPS extraction via heating, LPS purification with organic solvents, and treatment with proteinase K. LPSs that were extracted by using this method contained less than 2–3% protein and 1% total nucleic acid. We also demonstrated the structural integrity of the O-antigen and lipid A via the sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and matrix-assisted laser desorption ionization mass spectrometry (MALDI–MS) methods, respectively. We demonstrated the ability of the extracted LPSs to induce typical secretion of cytokines and chemokines by primary macrophages. Overall, this method may be used to isolate purified LPSs with preserved structures of both the O-antigen and lipid A and unchanged functional activity from small amounts of bacterial biomass.