3.4. Derivatization of N-glycans (see Note 7)

Fluorophores are conjugated at the reducing terminus of the glycan, which is uniquely constituted by a reactive carbonyl moiety. Many fluorophores are available including 2-aminobenzamide (2AB), AEAB and 2-aminopyridine (2AP or PA), the latter which is described here.

Transfer 80 µL of fresh 2-aminopyridine (PA) solution (see 2.1.4.1) to the glycan sample dried in a 1.5-ml polypropylene microcentrifuge tube, apply safety cap to avoid popping, and incubate in boiling water for 15 min.

Continue the reaction by transferring 4 µL of a fresh cyanoborohydride/PA solution (see 2.1.4.2) to the sample and incubating overnight at 90°C in an oven.

Dilute the sample in 1.5 ml of 0.5% acetic acid, apply to a 30 ml Sephadex G15 column (1 × 40 cm) equilibrated in 0.5% acetic acid, and collect 1.5 ml fractions. Transfer aliquots of fractions (80 µl) to a 96 F black plate and detect fluorescence in a microtiter plate reader (excitation/emission: 320/400nm). Pool fluorescent glycans eluting before the excess labeling reagent and lyophilize.