Figure 4.

Solid-phase synthesis of App1 oligosaccharides. A, schematic overview of the experimental setup. The acceptors are depicted in a simplified form. Cps1B-ΔTPR-His was immobilized on a HisTrap column, and the reaction mix containing the substrates (UDP-Gal and UDP-GlcNAc) and the acceptor oligosaccharides (pool I) was circulated for several consecutive rounds. B, Alcian blue/silver-stained PAGE (25%) of collected fractions (F = round of circulation). Pool I was used as marker material (because small fragments can diffuse from the gel, the lane was heavily overloaded to allow sufficient signal intensity). C, Western blot analysis of the loaded supernatant (S), collected fractions (F0–8), and eluted protein (E), stained against the C-terminal His tag of Cps1B-ΔTPR. No protein contamination could be detected in the reaction mixture throughout the experiment, indicating stable binding of Cps1B-ΔTPR to the column. After the experiment, Cps1B-ΔTPR could be eluted from the column using imidazole (E).