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 detection (1-10 ng/band) with zinc-imidazole followed by passive elution from 32 microm 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 simple replacement of water with other eluents would enhance the passive diffusion of LPS. We found that solutions of the detergents sodium dodecyl sulfate (SDS), sodium deoxycholate (DOC) and Triton X-100, or mixtures of the organic solvents acetonitrile and triethylamine and water, increased the recovery of a smooth LPS band from Vibrio cholerae O1 in a concentration-dependent manner. Furthermore, a quantitative recovery of rough or smooth LPS from V. cholerae O1, Escherichia coli O111:B4, E. coli K-235, or Serratia marcescens was feasible in 1% SDS or DOC after 3 h or in 5% triethylamine after only 2 min. A simple dilution of SDS or DOC or evaporation of triethylamine rendered the eluted LPS preparations compatible with biochemical activity determination, as tested by Limulus amebocyte lysate assay. Thus, this improved micropurification method may be a suitable interface between analytical gel electrophoresis and further characterization or use of LPS.
Pupo
E
E
Center for Genetic Engineering and Biotechnology, Havana, Cuba.
López
C M
CM
Alonso
M
M
Hardy
E
E
D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D007700:Kinetics; D008070:Lipopolysaccharides / Q000302:isolation & purification*; D016622:Silver Staining; D012997:Solvents; D014734:Vibrio cholerae / Q000737:chemistry*; D014867:Water
D003902:Detergents; D008070:Lipopolysaccharides; D012997:Solvents; D014867:Water