High-efficiency passive elution of bacterial lipopolysaccharides from polyacrylamide gels

We recently described a method for recovering polyacrylamide-gel-separated bacterial lipopolysaccharides (LPS) based on the sensitive on-gel LPS detec tion (1-10 ng/band) with zinc-imidazole followed by passive elution from 32 mu m average size gel microparticles into water. With this procedure, the recovery of rough- or semismooth-type LPS after 3 h elution is about 70-80% , while that of smooth LPS is only about 10%. Here we evaluated whether a s imple replacement of water with other eluents would enhance the passive dif fusion of LPS. We found that solutions of the detergents sodium dodecyl sul fate (SDS), sodium deoxycholate (DOC) and Triton X-100, or mixtures of the organic solvents acetonitrile and triethylamine and water, increased the re covery of a smooth LPS band from Vibrio cholerae O1 in a concentration-depe ndent manner. Furthermore, a quantitative recovery of rough or smooth LPS f rom V. cholerae O1, Escherichia coli O117:B4, E. coli K-235, or Serratia ma rcescens was feasible in 1% SDS or DOC after 3 h or in 5% triethylamine aft er only 2 min. A simple dilution of SDS or DOC or evaporation of triethylam ine rendered the eluted LPS preparations compatible with biochemical activi ty determination, as tested by Limulus amebocyte lysate assay. Thus, this i mproved micropurification method may be a suitable interface between analyt ical gel electrophoresis and further characterization or use of LPS.