Structures of SUPER TWIGs and their direct binding to Stx. (a) Structures of SUPER TWIG (0)3 (Mr = 2,006; trisaccharide = 1.50 × 10−3 mol/g), (1)6 (Mr = 4,001; trisaccharide = 1.50 × 10−3 mol/g), and (1)12 (Mr = 7,913; trisaccharide = 1.52 × 10−3 mol/g), carrying 3, 6, and 12 trisaccharides of Gb3, respectively. The numbers in parentheses indicate the generation numbers of the SUPER TWIGs. The zero and the first generation of the SUPER TWIGs have one and three linear silicon atoms, respectively, in their core structures. (b) TLC immunostaining assay. Double bands were detected, and both were confirmed to be Gb3 by anti-Gb3 staining (data not shown). The data are representative of three experiments. (c) Kinetic analysis of SUPER TWIG binding to immobilized Stx1-A2B5-GST by using the BIAcore system. The binding kinetics were analyzed by Scatchard plot to determine Kd and maximum binding values (means ± SE, n = 5). The maximum binding values of SUPER TWIG (0)3, (1)6, and (1)12 were 43 ± 2.9, 63 ± 2.6, and 57 ± 2.0 resonance units (RU), respectively.