Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) was performed on purified mNOTCH1 EGF1-18 and hTHBS1 TSR1-3 proteins (~100 ng) to biotinylate proteins modified with 6- or 7-Alk-Fuc. The reaction was performed with 100 µM Biotin picolyl azide (Click Chemistry Tools, Scottsdale, AZ, USA), 500 µM BTTP (3-(4-((bis((1-tertbutyl)-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)-propan-1-ol, a gift from Dr. Peng Wu) [62 (link)], 250 µM copper sulfate, 2.5 mM sodium ascorbate in water and gently shaking at room temperature for one hour. The biotinylated samples were then analyzed by Western blot. Samples were loaded onto 4–15% SDS-PAGE, transferred to nitrocellulose membrane and probed with anti-Myc antibody (Clone 9E10, Invitrogen, 1:2000), followed by IRDye 680-conjugated goat anti-mouse IgG antibody (LI-COR, 1:5000) to detect the total protein amount. To detect the biotinylated proteins, the nitrocellulose membrane was probed with IRDye 680RD streptavidin (LI-COR, 1:2000) or IRDye 800CW streptavidin (LI-COR, 1:2000). The Western blot bands were visualized using an Odyssey System (LI-COR).
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