Rapid isolation method for lipopolysaccharide and lipid A from Gram-negative bacteria

A fast, convenient extraction method for lipopolysaccharide (LPS), using a commercial RNA isolating reagent, allows the isolation of LPS or lipid A fr om low milligram (dry weight) quantities of bacterial cells. The method avo ids the use of specialized equipment and has been used for processing relat ively large numbers of samples. The major components of the commercial RNA isolating reagent, Tri-Reagent, are phenol and guanidinium thiocyanate in a queous solution. The bacterial cell membranes are disrupted with guanidiniu m thiocyanate, which eliminates the need for mechanical cell disruption (e. g. French press) or heating. LPS and its degradation products, with particu lar attention paid to its bioactive lipid A portion, were measured and comp ared with those from the most common conventional extraction method, hot ph enol-water. Negative ion quadrupole ion trap and matrix-assisted laser deso rption/ionization time-of-flight (MALDI-TOF) mass spectrometry, fatty acid composition analysis by capillary gas chromatography, total and free phosph ate by UV spectrophotometry and sodium dodecyl sulfate polyacrylamide gel e lectrophoresis (SDS-PAGE) showed that LPS and lipid A isolated using the Tr i-Reagent approach were cleaner and suffered less degradation through loss of phosphate and (or) fatty acyl side chains from lipid A. The Tri-Reagent extraction method generated low free phosphate contamination, 11% of the to tal phosphate concentration, whereas the hot phenol-water extraction method gave approximately 58% as free, inorganic phosphate. Similar results were observed for the degradation of fatty acyl side chains. The time required b y the new method is considerably shorter (two or three days) than that requ ired by conventional hot phenol-water extraction (about two weeks).