FAQ: What are the typical reaction conditions for O-Glycosidase?

Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate. Typical reaction conditions are as follows:
  1. Combine 10-20 µg of glycoprotein, 1 µL of 10X Glycoprotein Denaturing Buffer and H20 (if necessary) to make a 10 µL total reaction volume.
  2. Denature glycoprotein by heating reaction at 100°C for 10 minutes.
  3. Make a total reaction volume of 20 µL by adding 2 µL 10X GlycoBuffer 2, 2 µL 10% NP-40, 2 µL Neuraminidase, H20 and 1-5 µL O-Glycosidase.
  4. Incubate reaction at 37°C for 1 - 4 hours.
Note: O-Glycosidase can be used under either denaturing or native conditions. However, under denaturing conditions the enzyme activity is increased two-fold. This observation is substrate dependent. The reaction may be scaled-up linearly to accommodate large amounts of O-Glycosidase and larger reaction volumes.