Abstract
A simple and non-RI method for the identification and characterization of plant receptors with biotinylated ligands is presented. Biocytin hydrazide conjugate of N-acetylchitooctaose (GN8-Bio) was synthesized and used for the characterization of chitin elicitor binding protein, CEBiP. Membrane fractions were treated with GN8-Bio and cross-linked with glutaraldehyde. CEBiP was successfully biotinylated and detected both in the plasma membrane and microsomal membrane fractions by Western blotting with anti-biotin antibody followed by chemiluminescence detection. The binding characteristics of the GN8-Bio to the CEBiP showed a good agreement with the known specificity of CEBiP. The GN8-Bio-tagged CEBiP could also be purified from the membrane using an avidin column. These results indicated the usefulness of biotinylated ligands both for the purification and characterization of putative receptors or binding proteins for the ligands of interest.
