Biotinylation of Bacterial Lipopolysaccharide and Its Applications to Electron Microscopy
Abstract
We describe a procedure for lipopolysaccharide (lps) biotinylation using N-Biotinyl-l-lysine and application of the giotinylated LPS (Bi-LPS) to localization of LPS binding sites and subcellular distribution. Biotinylation of LPS was confirmed by enzyme-linked immunosorbent assay (ELISA), gel immunodiffusion, and immunodot techniques. The biological and toxicological activity of the Bi-LPS was tested by Limulus amoebocyte lysate (LAL) assays and histopathological examinations, respectively. Results showed that biotin was conjugated to LPS without disrupting the biological/toxicological activity of the molecule, which indicates that the biotin is directly linked to the polysaccharide portion of LPS. Localization of binding sites and subcellular distribution of Bi-LPS in human platelets and monocytes were studied by electron microscopy using an avidin-biotin-horseradish peroxidase (hrp) or streptavidin- gold method. Platelet surface were intensely stained by the reaction product of horseradish peroxidase (HPR) 5 min after incubation, and Bi-LPA provides a reliable, stable, and sensitive tool for determination of LPA binding sites and subcellular distribution. Keywords: LPS; Biotinylation; Lysine; Binding site; Subcellular distribution; Electron microscopy; Reprints; Biological Stains; Membranes biology.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jan 01, 1988
- Accession Number
- ADA202176
Entities
People
- Charles O. Odeyale
- Yuan-hsu Kang
Organizations
- Naval Medical Research Center