A sensitive method for the detection of Gram-negative bacterial lipopolysaccharide (LPS) blotted on nylon membranes is described. LPSs are separated by SDS-PAGE and then electrophoretically transferred to nylon membranes. Immobilized LPS is oxidized with periodate and then reacted with a hydrazide conjugated to the steroid, digoxigenin. LPS is visualized by alkaline phosphatase labelled antibodies against the steroid and the enzyme substrate 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium. LPS banding patterns of both rough (R-) and smooth (S-) type LPSs from over 15 different bacterial species are similar to those of silver stained companion gels, but without nonspecific staining of proteins. The detection of S-LPS from Pseudomonas aeruginosa F-D type 1 and R-LPS from Escherichia coli K12 is sensitive to 10-20 ng per lane. The use of this detection system in combination with antibody or lectin studies on identical blots can provide an effective tool in locating the precise position of certain epitopes or sequences in both R- and S-type LPSs on the blots.
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