Isolating glycoproteins using multilectin affinity columns

The multilectin column was prepared by mixing equal amount of agarose-bound Con A, agarose-bound WGA and agarose-bound Jacalin in an empty PD-10 disposable column (GE Healthcare, Piscataway, NJ, USA). The sample of 50 mL serum or plasma (Tab. 1) was diluted with multilectin column equilibrium buffer (20 mm Tris, 0.15 m NaCl, 1 mm Mn21, and 1 mm Ca21, pH 7.4) to a volume of 1 mL, and was loaded on a newly packed multilectin affinity column. After a 15 min reaction, the unbound proteins were eluted with 10 mL of equilibrium buffer, and the captured proteins were released with 12 mL of displacer solution (20 mm Tris, 0.5 m NaCl, 0.17 m methyl-a-d-mannopyranoside, 0.17 m N-acetyl-glucosamine, and 0.27 m galactose, pH 7.4). The multilectin affinity column captured fraction was collected and concentrated with 15 mL, 10 kDa Amicon filters (Millipore, Billerica, MA, USA).

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