Typical Reaction Conditions for α1-2, 3, 4, 6 Fucosidase (P0748)

Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate. Typical reaction conditions are as follows:
  1. Combine 1 μg of glycoprotein or 100 nM of oligosaccharide and H2O (if necessary) to make an 8 μl total reaction volume.
  2. Add 1 μl of 10X GlycoBuffer 1 and 1 μl of 10X BSA (diluted 1:10 from 100X concentration) to make a 10 μl total reaction volume.
  3. Add 1 μl of α1-2,3, 4,6 Fucosidase.
  4. Incubate at 37°C for 1 hour. 

Notes:
  • Reactions may be scaled-up linearly to accommodate larger reaction volumes. 
  • The amount of exoglycosidase enzyme required varies when different substrates are used. Start with 1-2 μl for 1ug of glycoprotein or 100 nM of oligosaccharide for one hour in a 10-25 μl reaction. If there is still undigested material, let the reaction go overnight.
  • α1-2,3,4,6 Fucosidase will partially cleave linear and branched α1-3 Fucose residues with low efficiency.
  • α1-2,3,4,6 Fucosidase will cleave branched α1-4 and α1-6 fucose residues but will not cleave branched α1-2 fucose residues. Longer incubation times (4 hours to overnight) may be needed for complex, branched oligosaccharide substrates.