O-Glycoprotease (IMPa) Non-Denaturing Reaction Protocol (NEB #P0761)

Optimal enzyme concentration and incubation times may vary for a particular substrate. Reactions may be scaled-up linearly to accommodate larger amounts of substrate and larger reaction volumes. Typical reaction conditions are as follows:

1. Combine 10 µg of glycoprotein or glycopeptide in 20mM Tris-HCl pH 8.0 to a total reaction volume of 50 μL.
   
   
2. Add 1 μL of O-Glycoprotease (IMPa), mix gently.
   
   
3. Incubate at 37˚C for 2 hours.
   
   Note - Some substrates may require extended incubation periods (up to 18 hours at 37°C).
   
   

Notes:

• Recommended substrate to enzyme ratio is 10 µg substrate to 1 µL enzyme.
  
  
• The optimal pH range for O-Glycoprotease (IMPa) is pH 7.0-8.0. Enzyme activity decreases significantly at pH values below 7.0 and above pH 8.0.
  
  
• Reactions can be filtered or used immediately for analysis by MS or MS/MS.
  
  
• Reactions can be stored frozen at -20°C for later use.