*4.6. Determination of TmC4-47.2 Toxin Binding Specificity to Carbohydrates*

The toxin was cut from 12% SDS-PAGE gel and eluted in PBS to identify toxin binding specificity using a competition assay performed according to Haab [55] using the DIG Glycan Differentiation Kit (#11210238001, Roche Applied Science, Germany). The pure control glycoproteins: carboxypeptidase Y, transferrin, fetuin, and asialofetuin or after pre-incubated with 1 μg of the toxin TmC4-47.2 for 1 h at 37 ◦C were administered in a 12% SDS–PAGE gel and transferred to a nitrocellulose membrane. Toxin/carbohydrate binding was revealed by incubation with different DIG-labeled lectins and alkaline phosphataseconjugated anti-DIG antibodies (Roche Molecular Biosciences). Characteristics of specific binding of lectins to carbohydrate moieties used to identify these structures in this study: GNA: *Galanthus nivalis* agglutinin specific to mannose: α(1–2), α(1–3), or α(1–6) linked to mannose; SNA: *Sambucus nigra* agglutinin specific to Sialic acid: α(2–6) to GalNAc; MAA: *Maackia amurensis* agglutinin specific to Sialic acid: α(2–3) to galactose N-linked α(2–3) to galactose O-linked; PNA: *Arachis hypogaea* Peanut agglutinin specific to Core and terminal galactose: Gal-β(1–3)-N-acetylgalactosamine; and DSA: *Datura stramonium* agglutinin specific to Core galactose: Gal-β(1–4)-N-acetylglucosamine.
