Typical Reaction Conditions for α1-2,3,6 Mannosidase (P0768)

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) in a total reaction volume of 8 μl.
  2. Add 1 μl of GlycoBuffer 4 (10X) and 1 μl of Zinc (10X) to make a 10 μl total reaction volume.
  3. Add 1 μl of α1-2,3,6 Mannosidase
  4. Incubate at 37°C for 1 hour

  • The optimal pH for α1-2,3,6 Mannosidase is 4.5. Enzyme activity is enhanced by the presence of 2 mM Zn2+ in the reaction.
  • In order to achieve complete removal of all non-reducing terminal α-linked mannose residues, it may be necessary to use an increased enzyme concentration as well as an overnight incubation time (18 hours).
  • To achieve complete removal of α1-6 mannose residues from a complex substrate, a sequential digest using α1-2,3,6 Mannosidase followed by α1-6 Mannosidase (NEB #P0727) may be necessary.
  • 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 1 μg 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.