Click Chemistry

EGCG-N₃ for the click chemistry was synthesized from EGCG and 6-azide-6-deoxyl-idose. The manuscript involving the detail of it's synthetic procedure is in preparation (Tanaka, H. et al., submitted for publication). HSA (1.0 mg/ml) was incubated with 1 mM EGCG-N₃ in PBS (pH 7.4) at 37°C. Click chemistry was performed using the reaction mixtures containing 1.0 mg/ml protein with 1 mM CuSO₄, 1 mM ascorbic acid, 0.1 mM tris((1-benzyl-1H-1,2,3- triazol-4-yl)methyl)amine (Anaspec, Inc., San Jose), and 20 μM alkyne-PEG4-biotin (Click Chemistry Tools). After incubation in the dark for 2 h at room temperature, the protein samples were boiled in the Laemmli sample buffer for 5 min, and the biotinylated proteins were then subjected to SDS-PAGE/Western blot followed by detection with HRP-conjugated NeutrAvidin and ECL.