Identification of Gangliosides as Bacterial Attachment Sites Using a TLC-Overlay Assay with Radioisotope-Labeled Bacteria.

This chapter describes a thin-layer chromatography (TLC) overlay assay for identifying gangliosides to which bacteria bind. In this method, gangliosides are first applied to a silica gel TLC plate and separated using an appropriate developing solvent. The TLC plate is then treated with polyisobutylmethacrylate to prevent nonspecific adsorption. Subsequently, the treated TLC plate is incubated with a PBS suspension of radioisotope-labeled bacteria for an appropriate duration. After incubation, unbound bacteria are washed away, and the TLC plate is analyzed using an imaging analyzer. The specificity of a ganglioside for binding to a particular bacterium is determined by quantifying the amount of radioisotope-labeled bacteria bound to the ganglioside on the TLC plate.